Antisense modulation of bcl-x expression

ABSTRACT

Compositions and methods are provided for modulating the expression of bcl-x. Antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding bcl-x are preferred. Methods of using these compounds for modulation of bcl-x expression and for treatment of diseases associated with expression of bcl-x are also provided. Methods of sensitizing cells to apoptotic stimuli are also provided.

[0001] The present application is a continuation of U.S. patentapplication Ser. No. 09/734,846 filed Dec. 12, 2000, which is acontinuation-in-part of U.S. patent application Ser. No. 09/323,743,filed Jun. 2, 1999, now issued as U.S. Pat. No. 6,214,986, which is acontinuation-in-part of U.S. patent application Ser. No. 09/277,020,filed Mar. 26, 1999, now issued as U.S. Pat. No. 6,210,892, which is acontinuation-in-part of U.S. patent application Ser. No. 09/167,921,filed Oct. 7, 1998, now issued as U.S. Pat. No. 6,172,216.

FIELD OF THE INVENTION

[0002] The present invention provides compositions and methods formodulating the expression of bcl-x and for inducing apoptosis. Inparticular, this invention relates to antisense compounds, particularlyoligonucleotides, specifically hybridizable with nucleic acids encodinghuman bcl-x. Such oligonucleotides have been shown to modulate theexpression of bcl-x.

BACKGROUND OF THE INVENTION

[0003] Programmed cell death, or apoptosis, is an essential feature ofgrowth and development, as the control of cell number is a balancebetween cell proliferation and cell death. Apoptosis is an active ratherthan a passive process, resulting in cell suicide as a result of any ofa number of external or internal signals. Apoptotic cell death ischaracterized by nuclear condensation, endonucleolytic degradation ofDNA at nucleosomal intervals (“laddering”) and plasma membrane blebbing.Programmed cell death plays an essential role in, for example, immunesystem development and nervous system development. In the former, Tcells displaying autoreactive antigen receptors are removed byapoptosis. In the latter, a significant reshaping of neural structuresoccurs, partly through apoptosis.

[0004] An increasing number of genes and gene products have beenimplicated in apoptosis. One of these is bcl-2, which is anintracellular membrane protein shown to block or delay apoptosis.Overexpression of bcl-2 has been shown to be related to hyperplasia,autoimmunity and resistance to apoptosis, including that induced bychemotherapy (Fang et al., J. Immunol. 1994, 153, 4388-4398). A familyof bcl-2-related genes has been described. All bcl-2 family membersshare two highly conserved domains, BH1 and BH2. These family membersinclude, but are not limited to, A-1, mcl-1, bax and bcl-x. Bcl-x wasisolated using a bcl-2 cDNA probe at low stringency due to its sequencehomology with bcl-2. Bcl-x was found to function as a bcl-2-independentregulator of apoptosis (Boise et al., Cell, 1993, 74, 597-608). Twoisoforms of bcl-x were reported in humans. Bcl-xl (long) contains thehighly conserved BH1 and BH2 domains. When transfected into an IL-3dependent cell line, bcl-xl inhibited apoptosis during growth factorwithdrawal in a manner similar to bcl-2. In contrast, the bcl-x shortisoform, bcl-xs, which is produced by alternative splicing and lacks a63-amino acid region of exon 1 containing the BH1 and BH2 domains,antagonizes the anti-apoptotic effect of either bcl-2 or bcl-xl. Asnumbered in Boise et al., Cell, 1993 74:, 597-608, the bcl-x transcriptcan be categorized into regions described by those of skill in the artas follows: nucleotides 1-134, 5′ untranslated region (5′-UTR);nucleotides 135-137, translation initiation codon (AUG); nucleotides135-836, coding region, of which 135-509 are the shorter exon 1 of thebcl-xs transcript and 135-698 are the longer exon 1 of the bcl-xltranscript; nucleotides 699-836, exon 2; nucleotides 834-836, stopcodon; and nucleotides 837-926, 3′ untranslated region (3′-UTR). Betweenexons 1 and 2 (between nucleotide 698 and 699) an intron is spliced outof the pre-mRNA when the mature bcl-xl (long) mRNA transcript isproduced. An alternative splice from position 509 to position 699produces the bcl-xs (short) mRNA transcript which is 189 nucleotidesshorter than the long transcript, encoding a protein product (bcl-xs)which is 63 amino acids shorter than bcl-xl. Thus nucleotide position698 is sometimes referred to in the art as the “5′ splice site” andposition 509 as the “cryptic 5′ splice site,” with nucleotide 699sometimes referred to as the “3′ splice site.”

[0005] Diseases and conditions in which apoptosis has been implicatedfall into two categories, those in which there is increased cellsurvival (i.e., apoptosis is reduced) and those in which there is excesscell death (i.e., apoptosis is increased). Diseases in which there is anexcessive accumulation of cells due to increased cell survival includecancer, autoimmune disorders and viral infections. Until recently, itwas thought that cytotoxic drugs killed target cells directly byinterfering with some life-maintaining function. However, of late, ithas been shown that exposure to several cytotoxic drugs with disparatemechanisms of action induces apoptosis in both malignant and normalcells. Manipulation of levels of trophic factors (e.g., by anti-estrogencompounds or those which reduce levels of various growth hormones) hasbeen one clinical approach to promote apoptosis, since deprivation oftrophic factors can induce apoptosis. Apoptosis is also essential forthe removal of potentially autoreactive lymphocytes during developmentand the removal of excess cells after the completion of an immune orinflammatory response. Recent work has clearly demonstrated thatimproper apoptosis may underlie the pathogenesis of autoimmune diseasesby allowing abnormal autoreactive lymphocytes to survive. For these andother conditions in which insufficient apoptosis is believed to beinvolved, promotion of apoptosis is desired. This can be achieved, forexample, by promoting cellular apoptosis or by increasing thesensitivity of cell to endogenous or exogenous apoptotic stimuli, forexample, cell signaling molecules such as TNF. or other cytokines,cytotoxic drugs or radiation. Promotion of or sensitization to apoptosisis believed to have clinical relevance in, for example, sensitizingcancer cells to chemotherapeutic drugs or radiation. It is also believedto be relevant in blocking angiogenesis which is necessary for tumorgrowth. This is because tumor cells release angiogenic factors torecruit angiogenic endothelial cells to the tumor site. It would bedesirable to sensitize these angiogenic endothelial cells to apoptoticstimuli (chemotherapeutic drugs, radiation, or endogenous TNF.) to blockangiogenesis and thus block tumor growth. Aberrant angiogenesis is alsoimplicated in numerous other conditions, for example maculardegeneration, diabetic retinopathy and retinopathy of prematurity, allof which can cause loss of vision. Aberrant angiogenesis is alsoimplicated in other, non-ocular conditions. Thus “aberrant” angiogenesiscan refer to excessive or insufficient angiogenesis, or undesiredangiogenesis (as, for example, in the case of angiogenesis whichsupports tumor growth. Blocking aberrant angiogenesis by sensitizingangiogenic endothelial cells to apoptotic stimuli is therefore desired.

[0006] In the second category, AIDS and neurodegenerative disorders likeAlzheimer's or Parkinson's disease represent disorders for which anexcess of cell death due to promotion of apoptosis (or unwantedapoptosis) has been implicated. Amyotrophic lateral sclerosis, retinitispigmentosa, and epilepsy are other neurologic disorders in whichapoptosis has been implicated. Apoptosis has been reported to occur inconditions characterized by ischemia, e.g. myocardial infarction andstroke. Apoptosis has also been implicated in a number of liverdisorders including obstructive jaundice and hepatic damage due totoxins and drugs. Apoptosis has also been identified as a key phenomenonin some diseases of the kidney, i.e. polycystic kidney, as well as indisorders of the pancreas including diabetes (Thatte, et al., Drugs,1997, 54, 511-532). For these and other diseases and conditions in whichunwanted apoptosis is believed to be involved, inhibitors of apoptosisare desired.

[0007] Antisense oligonucleotides have been used to elucidate the roleof several members of the bcl-2 family. Extensive studies usingantisense oligonucleotides targeted to bcl-2 have been performed, and anantisense compound (G3139, Genta, Inc.) targeted to human bcl-2 hasentered clinical trials for lymphoma and prostate cancer.

[0008] Amarante-Mendes et al., Oncogene, 1998, 16, 1383-1390, discloseantisense oligonucleotides targeted to bcr and bcl-x. The latterdownregulated the expression of bcl-xl and increased the susceptibilityof HL-60 Bcr-Abl cells to staurosporine.

[0009] U.S. Pat. No. 5,583,034 (Green et al.) discloses antisenseoligonucleotides which hybridize to the nucleic acid sequence of ananti-apoptotic gene, preferably to the translation start site ofbcr-abl.

[0010] Wang et al. used a phosphorothioate oligonucleotide targeted tothe bcl-x translation start site to block CD40L-mediated apoptoticrescue in murine WEHI-231 lymphoma cells (J. Immunol., 1995, 155,3722-3725).

[0011] Fujio et al. have used an antisense oligodeoxynucleotide targetedto murine and rat bcl-x mRNA to reduce bcl-xl protein expression (J.Clin. Invest., 1997, 99, 2898-2905). The compound tested was the same asthat of Wang et al. Oligonucleotide treatment inhibited thecytoprotective effect of leukemia inhibitory factor in mouse or ratcardiac myocytes.

[0012] Pollman et al. used antisense oligodeoxynucleotides withphosphorothioate backbones to downregulate bcl-xl expression in bloodvessel intimal cells (Nature Med., 1998, 4, 222-227). This resulted ininduction of apoptosis and regression of vascular lesions. Antisensesequences were targeted to the translation initiation codon ofmouse/human bcl-x (conserved sequence) and were used in rabbits. Gibbonset al., U.S. Pat. No. 5,776,905, disclose methods for targeted deletionof intimal lesion cells in the vasculature of a mammal with vasculardisease, preferably with antisense molecules specific for anti-apoptoticgenes, more preferably bcl-x and most preferably bcl-xl.

[0013] Thompson et al., U.S. Pat. No. 5,646,008 and WO 95/00642 describean isolated and purified polynucleotide that encodes a polypeptide otherthan bcl-2 that promotes or inhibits programmed vertebrate cell death.Preferably the polypeptide is bcl-xl, bcl-xs or bcl-x₁. Polypeptides,polynucleotides identical or complementary to a portion of the isolatedand purified polynucleotide, expression vectors, host cells, antibodiesand therapeutic and diagnostic methods of use are also provided.

[0014] Yang et al., WO 98/05777 disclose bcl-x. (gamma), a novel isoformof the bcl-x family which includes an ankyrin domain. Polypeptide andnucleic acid sequences for this isoform are disclosed, as well as, interalia, methods for modulating bcl-x. activity, including antisensemethods.

SUMMARY OF THE INVENTION

[0015] The present invention is directed to antisense compounds,particularly oligonucleotides, which are targeted to a nucleic acidencoding bcl-x, and which modulate the expression of bcl-x.

[0016] One embodiment of the present invention is an antisense compound8 to 30 nucleobases in length targeted to a nucleic acid moleculeencoding a human bcl-x which modulates the expression of human bcl-x.Preferably, the antisense compound is an antisense oligonucleotide. Inone aspect of this preferred embodiment, the antisense oligonucleotidecomprises at least one modified internucleoside linkage. Advantageously,the modified internucleoside linkage is a phosphorothioate, morpholinoor peptide-nucleic acid linkage. Preferably, the antisenseoligonucleotide comprises at least one modified sugar moiety. In oneaspect of this preferred embodiment, the modified sugar moiety is a2′-O-methoxyethyl or a 2′-dimethylaminooxyethoxy sugar moiety.Advantageously, substantially all sugar moieties of the antisenseoligonucleotide are modified sugar moieties. Preferably, the antisenseoligonucleotide comprises at least one modified nucleobase. In oneaspect of this preferred embodiment, the modified is a 5-methylcytosine.Preferably, each 2′-O-methoxyethyl modified cytosine nucleobase is a5-methylcytosine. In another aspect of this preferred embodiment, theantisense compound is a chimeric oligonucleotide.

[0017] The present invention also provides a pharmaceutical compositioncomprising the antisense compound described above and a pharmaceuticallyacceptable carrier or diluent. The pharmaceutical composition mayfurther comprise a colloidal dispersion system. Preferably, theantisense compound is an antisense oligonucleotide. In one aspect ofthis preferred embodiment, the antisense compound is targeted to bcl-xl.Preferably, the antisense compound is targeted to bcl-xl andpreferentially inhibits the expression of bcl-xl. In another aspect, theantisense compound is targeted to a region of a nucleic acid moleculeencoding bcl-xl which is not found in a nucleic acid molecule encodingbcl-xs. Advantageously, the antisense compound promotes apoptosis.Preferably, the antisense compound is targeted to a region of a nucleicacid molecule encoding bcl-xs and reduces the expression of bcl-xs. Inanother aspect, the antisense compound inhibits apoptosis. The antisensecompound may also alter the ratio of bcl-x isoforms expressed by a cellor tissue. Preferably, the antisense compound increases the ratio ofbcl-xl to bcl-xs expressed. Alternatively, the antisense compounddecreases the ratio of bcl-xl to bcl-xs expressed.

[0018] Another embodiment of the present invention is a method ofinhibiting the expression of bcl-x in human cells or tissues comprisingcontacting the cells or tissues with the antisense compound describedabove so that expression of bcl-x is inhibited.

[0019] The present invention also provides a method of treating ananimal having a disease or condition associated with bcl-x comprisingadministering to the animal a therapeutically or prophylacticallyeffective amount of the antisense compound described above so thatexpression of bcl-x is inhibited.

[0020] Another embodiment of the present invention is a method oftreating an animal having a disease or condition characterized by areduction in apoptosis comprising administering to said animal aprophylactically or therapeutically effective amount of the antisensecompound described above. Preferably, the antisense compound is targetedto a nucleic acid molecule encoding bcl-xl and preferentially inhibitsthe expression of bcl-xl. pharmaceutical composition may furthercomprise a chemotherapeutic agent.

[0021] The present invention also provides a method of treating cancerin an animal comprising: (a) administering to the animal thepharmaceutical composition described above; and (b) administering to theanimal a chemotherapeutic agent.

[0022] Another embodiment of the invention is a method of sensitizing acell to an apoptotic stimulus comprising treating the cell with thecomposition described above. Preferably, the apoptotic stimulus isradiation; more preferably, ultraviolet radiation. Alternatively, theapoptotic stimulus is a cancer chemotherapeutic drug. Preferably, thecancer chemotherapeutic drug is VP-16, cisplatinum or taxol. In anotheraspect of this preferred embodiment, the apoptotic stimulus is acellular signaling molecule. Preferably, the apoptotic stimulus isceramide, a cytokine or staurosporine. In one aspect of this preferredembodiment, the apoptotic stimulus causes mitochondrial dysfunction.Advantageously, the mitochondrial dysfunction is loss of mitochondrialmembrane potential. Preferably, the cell is a cancer cell. In one aspectof this preferred embodiment, the cancer cell is a glioblastoma orleukemia cell.

[0023] The present invention also provides a method of promotingapoptosis of cancer cells, comprising contacting the cells with theantisense compound described above. The method may further comprise thestep of contacting the cells with a chemotherapeutic agent. Preferably,chemotherapeutic agent is doxorubicin or dexamethasone. In one aspect ofthis preferred embodiment, the cancer cells are glioblastoma cells orleukemia cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 is a graph which shows bcl-xl mRNA levels in the Shionogimouse tumor model. Shionogi tumors were harvested before and at severaltimes after castration. Poly A+ RNA was extracted from each tumor tissueand then analyzed for bcl-xl and G3PDH levels by Northern blottingfollowed by quantitation with a laser densitometer. Each columnrepresents the mean value with standard deviation.

[0025]FIG. 2 is a graph which shows bcl-xl mRNA levels afternormalization to G3PDH mRNA levels in Shionogi tumor cells followingtreatment with various concentrations of antisense bcl-xl or mismatchcontrol oligodeoxynucleotide. Each point represents the mean oftriplicate analysis with standard deviation. *: significantly differentfrom mismatch control ODN treatment: p<0.01 (Student's t-test).

[0026]FIG. 3 is a graph which shows the effects of antisense bcl-xloligodeoxynucleotide ISIS 16009 administration on Shionogi tumor growth.Beginning 1 day postcastration, 12.5 mg/kg ISIS 16009 or control ODN#1was injected i.p. once daily for 40 days into each mouse bearingShionogi tumors. Tumor volume was measured twice weekly and calculatedby the formula length×width×depth×0.5236. Each point represents the meantumor volume in each experimental group containing 7 mice with standarddeviation. * and **: significantly different from control ODN#1treatment; p<0.05 and 0.01, respectively (Student's t-test).

[0027]FIG. 4 is a graph which shows bcl-xl mRNA levels afternormalization to G3PDH mRNA levels in Shionogi tumors followingtreatment with ISIS 16009 or control ODN#1. Quantitation was performedwith a laser densitometer. Each column represents the mean value withstandard deviation. *: significantly different from control ODN#1treatment; p<0.01 (Student's t-test).

[0028]FIG. 5 is a graph showing viability analysis of M059K humanglioblastoma cells treated with taxol (2.5 nM) and oligonucleotides.Taxol was added to all groups at day 2. The effect of viability in thebcl-xl antisense (ISIS 16009) (A), mismatch oligodeoxynucleotide (M) andsaline (S) groups is shown as relative viability compared to cellstreated with saline only.

[0029]FIG. 6 shows a FACS analysis of MO59K cells treated with taxol(2.5 nM) and ISIS 16009 or mismatch oligonucleotides. Bcl-xl antisenseoligonucleotide treatment increased sub-G0-G1 DNA content *thick line)and reduced the number of cells in G0/G1 phase compared to mismatchtreated cells (thin line), suggesting that the increased apoptosisinduced by down-regulation of bcl-xl is cell-cycle specific.

[0030] FIGS. 7A-B show the dose dependence of bcl-x antisense effects onCEM cell survival (FIG. 7A) and bcl-x protein levels (FIG. 7B). CEMcells were electroporated with the indicated concentration of bcl-xantisense or control scrambled oligonucleotide. Twenty hours later, thenumber of viable cells was counted by propidium iodide exclusion andflow cytometry. Each value is the average of three differentdeterminations within the same experiment. The error bars indicate onestandard deviation. The data is representative of three differentexperiments with similar results.

[0031]FIG. 8 shows the effect of bcl-x antisense oligonucleotide on CEMcell growth. After electroporation with bcl-x antisense or controloligonucleotide at 20 μM, CEM cells were cultured for 20 hours, washedand replated at 10⁵/ml. At the indicated hours after electroporation,the number of viable cells was determined by propidium iodide exclusionand flow cytometry.

[0032] FIGS. 9A-B show the effect of bcl-x antisense oligonucleotide onCEM cell sensitivity to doxorubicin (FIG. 9A) and dexamethasone (FIG.9B). After electroporation with bcl-x antisense or controloligonucleotide at 20 μM, CEM cells were cultured for 20 hours, washedand replated at 10⁵/ml with the indicated concentration of drug. After24 or 48 hours, viable cell counts were determined by propidium iodideexclusion and flow cytometry. The data is representative of threedifferent experiments with similar results.

[0033]FIG. 10 shows the combination index (CI) plot displaying thesynergistic effect for the combination of bcl-x antisense with eitherdoxorubicin or dexamethasone. The data used for the calculations areshown in FIG. 9. The classifications of the extent of synergy are asdefined by Chou et al. (“The median-effect principle and the combinationindex for quantification of synergism and antagonism. In Synergism andAntagonism in Chemotherapy, T.-C. Chou and D. Rideout, eds., AcademicPress, New York, pp. 61-102).

[0034]FIG. 11 shows that reduction of bcl-xl expression sensitizes miceto fas antibody-induced death.

DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention comprehends antisense compounds capable ofmodulating expression of human bcl-x and of its isoforms, bcl-xl andbcl-xs. Bcl-xl inhibits apoptosis and therefore inhibitors of bcl-xl,particularly specific inhibitors of bcl-xl such as the antisensecompounds of the present invention, are desired as promoters ofapoptosis. In contrast, the bcl-x short isoform, bcl-xs, antagonizes theanti-apoptotic effect and therefore promotes apoptosis. Inhibitors ofbcl-xs are desired as inhibitors of apoptosis. Antisense compounds whichspecifically inhibit the expression of a particular isoform, eitherbcl-xl or bcl-xs, of bcl-x, or which alter the expression ratio of thesetwo isoforms, are particularly useful for both research and therapeutic,including prophylactic, uses.

[0036] The present invention employs oligomeric antisense compounds,particularly oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding bcl-x, ultimately modulating the amountof bcl-x produced. This is accomplished by providing antisense compoundswhich specifically hybridize with one or more nucleic acids encodingbcl-x. As used herein, the terms “target nucleic acid” and “nucleic acidencoding bcl-x” encompass DNA encoding bcl-x, RNA (including pre-mRNAand mRNA) transcribed from such DNA, and also cDNA derived from suchRNA. The specific hybridization of an oligomeric compound with itstarget nucleic acid interferes with the normal function of the nucleicacid. This modulation of function of a target nucleic acid by compoundswhich specifically hybridize to it is generally referred to as“antisense”. The functions of DNA to be interfered with includereplication and transcription. The functions of RNA to be interferedwith include all vital functions such as, for example, translocation ofthe RNA to the site of protein translation, translation of protein fromthe RNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity which may be engaged in or facilitated by the RNA.The overall effect of such interference with target nucleic acidfunction is modulation of the expression of bcl-x. In the context of thepresent invention, “modulation” means either an increase (stimulation)or a decrease (inhibition) in the expression of a gene product. In thecontext of the present invention, inhibition is a preferred form ofmodulation of gene expression and mRNA is a preferred target. Further,since many genes (including bcl-x) have multiple transcripts,“modulation” also includes an alteration in the ratio between geneproducts, such as alteration of mRNA splice products.

[0037] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding bcl-x. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding bcl-x, regardless of the sequence(s) of such codons.

[0038] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0039] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0040] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. It has also beenfound that introns can also be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0041] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0042] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, or in the case of in vitro assays, under conditions in whichthe assays are performed.

[0043] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0044] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotides have been safely and effectively administered to humansand numerous clinical trials are presently underway. It is thusestablished that oligonucleotides can be useful therapeutic modalitiesthat can be configured to be useful in treatment regimes of cells,tissues and animals, especially humans. In the context of thisinvention, the term “oligonucleotide” refers to an oligomer or polymerof ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimeticsthereof. This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0045] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about30 nucleobases. Particularly preferred are antisense oligonucleotidescomprising from about 8 to about 30 nucleobases (i.e. from about 8 toabout 30 linked nucleosides). As is known in the art, a nucleoside is abase-sugar combination. The base portion of the nucleoside is normally aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. Nucleotides are nucleosidesthat further include a phosphate group covalently linked to the sugarportion of the nucleoside. For those nucleosides that include apentofuranosyl sugar, the phosphate group can be linked to either the2′-, 3′- or 5′-hydroxyl moiety of the sugar. In formingoligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound. In turnthe respective ends of this linear polymeric structure can be furtherjoined to form a circular structure. However, open linear structures aregenerally preferred. Within the oligonucleotide structure, the phosphategroups are commonly referred to as forming the internucleoside backboneof the oligonucleotide. The normal linkage or backbone of RNA and DNA isa 3′ to 5′ phosphodiester linkage.

[0046] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0047] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acidforms are also included.

[0048] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; and 5,625,050, each of which is hereinincorporated by reference.

[0049] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

[0050] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

[0051] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0052] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0053] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br,CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes an alkoxyalkoxy group, 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78, 486-504). A further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE.

[0054] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference.

[0055] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in The Concise Encyclopedia Of PolymerScience And Engineering, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandteChemie, International Edition, 1991, 30, 613, and those disclosed bySanghvi, Y. S., Crooke, S. T., and Lebleu, B. eds., Antisense Researchand Applications, CRC Press, Boca Raton, 1993, pp. 289-302. Certain ofthese nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2□ C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

[0056] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121;5,596,091; 5,614,617; 5,681,941; and 5,750,692, each of which is hereinincorporated by reference.

[0057] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid(Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol orundecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118;Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al.,Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

[0058] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,each of which is herein incorporated by reference.

[0059] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Cleavage of the RNAtarget can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0060] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, each of which is herein incorporated by reference.

[0061] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0062] The antisense compounds of the invention are synthesized in vitroand do not include antisense compositions of biological origin, orgenetic vector constructs designed to direct the in vivo synthesis ofantisense molecules.

[0063] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes, receptortargeted molecules, oral, rectal, topical or other formulations, forassisting in uptake, distribution and/or absorption. RepresentativeUnited States patents that teach the preparation of such uptake,distribution and/or absorption assisting formulations include, but arenot limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0064] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0065] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl)phosphate] derivatives according to the methodsdisclosed in WO 93/24510 or in WO 94/26764.

[0066] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0067] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred addition saltsare acid salts such as the hydrochlorides, acetates, salicylates,nitrates and phosphates. Other suitable pharmaceutically acceptablesalts are well known to those skilled in the art and include basic saltsof a variety of inorganic and organic acids, such as, for example, withinorganic acids, such as for example hydrochloric acid, hydrobromicacid, sulfuric acid or phosphoric acid; with organic carboxylic,sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, forexample acetic acid, propionic acid, glycolic acid, succinic acid,maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malicacid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaricacid, glucuronic acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoicacid, 2-acetoxybenzoic acid, embolic acid, nicotinic acid orisonicotinic acid; and with amino acids, such as the 20 alpha-aminoacids involved in the synthesis of proteins in nature, for exampleglutamic acid or aspartic acid, and also with phenylacetic acid,methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid,ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfoicacid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2-or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid(with the formation of cyclamates), or with other acid organiccompounds, such as ascorbic acid. Pharmaceutically acceptable salts ofcompounds may also be prepared with a pharmaceutically acceptablecation. Suitable pharmaceutically acceptable cations are well known tothose skilled in the art and include alkaline, alkaline earth, ammoniumand quaternary ammonium cations. Carbonates or hydrogen carbonates arealso possible.

[0068] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0069] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of bcl-x is treated by administering antisense compounds inaccordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

[0070] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding bcl-x, enabling sandwich and other assays to easily beconstructed to exploit this fact. Hybridization of the antisenseoligonucleotides of the invention with a nucleic acid encoding bcl-x canbe detected by means known in the art. Such means may includeconjugation of an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection means. Kits using suchdetection means for detecting the level of bcl-x in a sample may also beprepared.

[0071] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0072] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful.

[0073] Compositions and formulations for oral administration includepowders or granules, suspensions or solutions in water or non-aqueousmedia, capsules, sachets or tablets. Thickeners, flavoring agents,diluents, emulsifiers, dispersing aids or binders may be desirable.

[0074] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0075] Pharmaceutical compositions and/or formulations comprising theoligonucleotides of the present invention may also include penetrationenhancers in order to enhance the alimentary delivery of theoligonucleotides. Penetration enhancers may be classified as belongingto one of five broad categories, i.e., fatty acids, bile salts,chelating agents, surfactants and non-surfactants (Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, 8, 91-192; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33).One or more penetration enhancers from one or more of these broadcategories may be included.

[0076] Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid,linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a.1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arichidonic acid,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, mono- and di-glycerides and physiologically acceptablesalts thereof (i.e., oleate, laurate, caprate, myristate, palmitate,stearate, linoleate, etc.) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, 8:2, 91-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7:1, 1-33; El-Hariri et al., J.Pharm. Pharmacol., 1992, 44, 651-654). Examples of some presentlypreferred fatty acids are sodium caprate and sodium laurate, used singlyor in combination at concentrations of 0.5 to 5%.

[0077] The physiological roles of bile include the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 In: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York,N.Y., 1996, pages 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus, the term“bile salt” includes any of the naturally occurring components of bileas well as any of their synthetic derivatives. A presently preferredbile salt is chenodeoxycholic acid (CDCA) (Sigma Chemical Company, St.Louis, Mo.), generally used at concentrations of 0.5 to 2%.

[0078] Complex formulations comprising one or more penetration enhancersmay be used. For example, bile salts may be used in combination withfatty acids to make complex formulations. Preferred combinations includeCDCA combined with sodium caprate or sodium laurate (generally 0.5 to5%).

[0079] Chelating agents include, but are not limited to, disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, 8:2, 92-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7:1, 1-33; Buur et al., J.Control Rel., 1990, 14, 43-51). Chelating agents have the addedadvantage of also serving as DNase inhibitors.

[0080] Surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8:2,92-191); and perfluorochemical emulsions, such as FC-43 (Takahashi etal., J. Pharm. Pharmacol., 1988, 40, 252-257).

[0081] Non-surfactants include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8:2,92-191); and non-steroidal anti-inflammatory agents such as diclofenacsodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm.Pharmacol., 1987, 39, 621-626).

[0082] As used herein, “carrier compound” refers to a nucleic acid, oranalog thereof, which is inert (i.e., does not possess biologicalactivity per se) but is recognized as a nucleic acid by in vivoprocesses that reduce the bioavailability of a nucleic acid havingbiological activity by, for example, degrading the biologically activenucleic acid or promoting its removal from circulation. Thecoadministration of a nucleic acid and a carrier compound, typicallywith an excess of the latter substance, can result in a substantialreduction of the amount of nucleic acid recovered in the liver, kidneyor other extracirculatory reservoirs, presumably due to competitionbetween the carrier compound and the nucleic acid for a common receptor.For example, the recovery of a partially phosphorothioatedoligonucleotide in hepatic tissue is reduced when it is coadministeredwith polyinosinic acid, dextran sulfate, polycytidic acid or4-acetamido-4′-isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0083] In contrast to a carrier compound, a “pharmaceutically acceptablecarrier” (excipient) is a pharmaceutically acceptable solvent,suspending agent or any other pharmacologically inert vehicle fordelivering one or more nucleic acids to an animal. The pharmaceuticallyacceptable carrier may be liquid or solid and is selected with theplanned manner of administration in mind so as to provide for thedesired bulk, consistency, etc., when combined with a nucleic acid andthe other components of a given pharmaceutical composition. Typicalpharmaceutically acceptable carriers include, but are not limited to,binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose andother sugars, microcrystalline cellulose, pectin, gelatin, calciumsulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate,etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidalsilicon dioxide, stearic acid, metallic stearates, hydrogenatedvegetable oils, corn starch, polyethylene glycols, sodium benzoate,sodium acetate, etc.); disintegrates (e.g., starch, sodium starchglycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate,etc.). Sustained release oral delivery systems and/or enteric coatingsfor orally administered dosage forms are described in U.S. Pat. Nos.4,704,295; 4,556,552; 4,309,406; and 4,309,404.

[0084] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional compatiblepharmaceutically-active materials such as, e.g., antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the composition of present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the invention.

[0085] Regardless of the method by which the antisense compounds of theinvention are introduced into a patient, colloidal dispersion systemsmay be used as delivery vehicles to enhance the in vivo stability of thecompounds and/or to target the compounds to a particular organ, tissueor cell type. Colloidal dispersion systems include, but are not limitedto, macromolecule complexes, nanocapsules, microspheres, beads andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, liposomes and lipid:oligonucleotide complexes ofuncharacterized structure. A preferred colloidal dispersion system is aplurality of liposomes. Liposomes are microscopic spheres having anaqueous core surrounded by one or more outer layer(s) made up of lipidsarranged in a bilayer configuration (see, generally, Chonn et al.,Current Op. Biotech., 1995, 6, 698-708).

[0086] Certain embodiments of the invention provide for liposomes andother compositions containing (a) one or more antisense compounds and(b) one or more other chemotherapeutic agents which function by anon-antisense mechanism. Examples of such chemotherapeutic agentsinclude, but are not limited to, anticancer drugs such as daunorubicin,dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard,chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine(5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine,etoposide, teniposide, cisplatin and diethylstilbestrol (DES). See,generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkowet al., eds., 1987, Rahway, N.J., pp. 1206-1228. Anti-inflammatorydrugs, including but not limited to nonsteroidal anti-inflammatory drugsand corticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pp. 2499-2506 and 46-49, respectively. Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

[0087] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Two or more combinedcompounds may be used together or sequentially.

[0088] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0089] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

[0090] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxyand 2′-alkoxy Amidites

[0091] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylPhosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham, Mass. or Glen Research, Inc. Sterling, Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, the standard cycle for unmodifiedoligonucleotides was utilized, except the wait step after pulse deliveryof tetrazole and base was increased to 360 seconds.

[0092] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me—C)nucleotides were synthesized according to published methods (Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

[0093] 2′-Fluoro Amidites

[0094] 2′-Fluorodeoxyadenosine Amidites 2′-fluoro oligonucleotides weresynthesized as described previously by Kawasaki, et. al., J. Med. Chem.,1993, 36, 831-841 and U.S. Pat. No. 5,670,633, herein incorporated byreference. Briefly, the protected nucleosideN6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizingcommercially available 9-beta-D-arabinofuranosyladenine as startingmaterial and by modifying literature procedures whereby the2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladeninewas selectively protected in moderate yield as the3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups was accomplished using standard methodologies andstandard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

[0095] 2′-Fluorodeoxyguanosine

[0096] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0097] 2′-Fluorouridine

[0098] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

[0099] 2′-Fluorodeoxycytidine

[0100] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0101] 2′-O-(2-Methoxyethyl) Modified Amidites

[0102] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

[0103] 2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

[0104] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60□C. at 1 mmHg for 24 hours) to give a solid that was crushed to a light tan powder(57 g, 85% crude yield). The NMR spectrum was consistent with thestructure, contaminated with phenol as its sodium salt (ca. 5%). Thematerial was used as is for further reactions or purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a white solid, mp 222-4□C.

[0105] 2′-O-Methoxyethyl-5-methyluridine

[0106] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160□C. After heating for 48 hours at 155-160□C.,the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/Acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product. Additional material was obtained byreworking impure fractions.

[0107] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0108] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/Hexane/Acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 gof product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

[0109]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0110] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by tlc by first quenching the tlc sample with the additionof MeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35□C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

[0111]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0112] A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5□C. and stirred for 0.5 hours using an overhead stirrer.POCl₃ was added dropwise, over a 30 minute period, to the stirredsolution maintained at 0-10□C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

[0113] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0114] A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100□C. for 2 hours (tlc showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

[0115]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0116] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, tlc showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/Hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

[0117]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0118]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tlc showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

Example 2

[0119] Oligonucleotide Synthesis

[0120] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 380B) using standard phosphoramidite chemistrywith oxidation by iodine.

[0121] Phosphorothioates (P═S) are synthesized as per the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 seconds and was followed by the capping step.After cleavage from the CPG column and deblocking in concentratedammonium hydroxide at 55□C. (18 hr), the oligonucleotides were purifiedby precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

[0122] Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

[0123] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0124] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0125] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0126] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0127] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0128] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0129] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3

[0130] Oligonucleoside Synthesis

[0131] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0132] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0133] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0134] PNA Synthesis

[0135] Peptide nucleic acids (PNAs) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

[0136] Synthesis of Chimeric Oligonucleotides

[0137] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0138] [2′-O-Me]—[2′-deoxy]—[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

[0139] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2′-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 Ammonia/Ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hours at roomtemperature is then done to deprotect all bases and sample was againlyophilized to dryness. The pellet is resuspended in 1M TBAF in THF for24 hours at room temperature to deprotect the 2′ positions. The reactionis then quenched with 1M TEAA and the sample is then reduced to ½ volumeby rotovac before being desalted on a G25 size exclusion column. Theoligo recovered is then analyzed spectrophotometrically for yield andfor purity by capillary electrophoresis and by mass spectrometry.

[0140] [2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[0141] [2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxy-ethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl)amidites for the 2′-O-methylamidites.

[0142] [2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxyPhosphorothioate]—[2′-O-(2-Methoxyethyl)Phosphodiester] ChimericOligonucleotides

[0143] [2′-O-(2-methoxyethyl phosphodiester]—[2′-deoxyphosphorothioate]—[2′-O-(methoxyethyl)phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0144] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0145] Oligonucleotide Isolation

[0146] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55□C.for 18 hours, the oligonucleotides or oligonucleosides were purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

[0147] Oligonucleotide Synthesis—96 Well Plate Format

[0148] Oligonucleotides are synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a standard 96 well format.Phosphodiester internucleotide linkages are afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages are generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites are purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

[0149] Oligonucleotides are cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60□C.) for 12-16 hoursand the released product then dried in vacuo. The dried product is thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

[0150] Oligonucleotide Analysis—96 Well Plate Format

[0151] The concentration of oligonucleotide in each well is assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products is evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition isconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates are diluted from the master plateusing single and multi-channel robotic pipettors. Plates are judged tobe acceptable if at least 85% of the compounds on the plate are at least85% full length.

Example 9

[0152] Cell Culture and Oligonucleotide Treatment

[0153] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR, RNAse protection assay(RPA) or Northern blot analysis. The following four human cell types areprovided for illustrative purposes, but other cell types can beroutinely used.

[0154] T-24 Cells

[0155] The transitional cell bladder carcinoma cell line T-24 isobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells are routinely cultured in complete McCoy's 5A basalmedia (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells are routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells are seeded into 96-well plates (Falcon-Primaria #3872)at a density of 7000 cells/well for use in RT-PCR analysis.

[0156] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0157] A549 Cells:

[0158] The human lung carcinoma cell line A549 is obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells areroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells are routinely passaged by trypsinization anddilution when they reached 90% confluence.

[0159] NHDF Cells:

[0160] Human neonatal dermal fibroblast (NHDF) are obtained from theClonetics Corporation (Walkersville Md.). NHDFs are routinely maintainedin Fibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells are maintained for upto 10 passages as recommended by the supplier.

[0161] HEK Cells:

[0162] Human embryonic keratinocytes (HEK) are obtained from theClonetics Corporation (Walkersville Md.). HEKs are routinely maintainedin Keratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells are routinelymaintained for up to 10 passages as recommended by the supplier.

[0163] Treatment with Antisense Compounds

[0164] When cells reached 80% confluency, they are treated witholigonucleotide. For cells grown in 96-well plates, wells are washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired oligonucleotide at a final concentration of150 nM. After 4 hours of treatment, the medium is replaced with freshmedium. Cells are harvested 16 hours after oligonucleotide treatment.

Example 10

[0165] Analysis of Oligonucleotide Inhibition of bcl-x Expression

[0166] Antisense modulation of bcl-x expression can be assayed in avariety of ways known in the art. For example, bcl-x mRNA levels can bequantitated by Northern blot analysis, RNAse protection assay (RPA),competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR).RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.Methods of RNA isolation are taught in, for example, Ausubel, et al.,Current Protocols in Molecular Biology, Volume 1, John Wiley & Sons,Inc., 1993, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3. Northern blot analysis isroutine in the art and is taught in, for example, Ausubel, et al.,Current Protocols in Molecular Biology, Volume 1, John Wiley & Sons,Inc., 1996, pp. 4.2.1-4.2.9. Real-time quantitative (PCR) can beconveniently accomplished using the commercially available ABI PRISM™7700 Sequence Detection System, available from PE-Applied Biosystems,Foster City, Calif. and used according to manufacturer's instructions.Other methods of PCR are also known in the art.

[0167] Bcl-x protein levels can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), ELISA, flow cytometry or fluorescence-activated cellsorting (FACS). Antibodies directed to bcl-x can be identified andobtained from a variety of sources, such as PharMingen Inc., San DiegoCalif., or can be prepared via conventional antibody generation methods.Methods for preparation of polyclonal antisera are taught in, forexample, Ausubel, et al., Current Protocols in Molecular Biology, Volume2, John Wiley & Sons, Inc., 1997, pp. 11.12.1-11.12.9. Preparation ofmonoclonal antibodies is taught in, for example, Ausubel, et al.,Current Protocols in Molecular Biology, Volume 2, John Wiley & Sons,Inc., 1997, pp. 11.4.1-11.11.5.

[0168] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, et al., Current Protocols in MolecularBiology, Volume 2, John Wiley & Sons, Inc., 1998, pp. 10.16.1-10.16.11.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, et al., Current Protocols in MolecularBiology, Volume 2, John Wiley & Sons, Inc., 1997, pp. 10.8.1-10.8.21.Enzyme-linked immunosorbent assays (ELISA) are standard in the art andcan be found at, for example, Ausubel, et al., Current Protocols inMolecular Biology, Volume 2, John Wiley & Sons, Inc., 1991, pp.11.2.1-11.2.22.

Example 11

[0169] Poly(A)+ mRNA Isolation

[0170] Poly(A)+ mRNA is isolated according to Miura et al., Clin. Chem.,1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolation aretaught in, for example, Ausubel, et al., Current Protocols in MolecularBiology, Volume 1, John Wiley & Sons, Inc., 1993, pp. 4.5.1-4.5.3.Briefly, for cells grown on 96-well plates, growth medium is removedfrom the cells and each well is washed with 200 μL cold PBS. 60 μL lysisbuffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mMvanadyl-ribonucleoside complex) is added to each well, the plate isgently agitated and then incubated at room temperature for five minutes.55 μL of lysate is transferred to Oligo d(T) coated 96-well plates (AGCTInc., Irvine Calif.). Plates are incubated for 60 minutes at roomtemperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HClpH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate isblotted on paper towels to remove excess wash buffer and then air-driedfor 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheatedto 70□C. is added to each well, the plate is incubated on a 90□C. hotplate for 5 minutes, and the eluate is then transferred to a fresh96-well plate.

[0171] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12

[0172] Total RNA Isolation

[0173] Total mRNA is isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium is removed from the cells and each well iswashed with 200 μL cold PBS. 100 μL Buffer RLT is added to each well andthe plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol isthen added to each well and the contents mixed by pipetting three timesup and down. The samples are then transferred to the RNEASY 96™ wellplate attached to a QIAVAC™ manifold fitted with a waste collection trayand attached to a vacuum source. Vacuum is applied for 15 seconds. 1 mLof Buffer RW1 is added to each well of the RNEASY 96™ plate and thevacuum again applied for 15 seconds. 1 mL of Buffer RPE is then added toeach well of the RNEASY 96™ plate and the vacuum applied for a period of15 seconds. The Buffer RPE wash is then repeated and the vacuum isapplied for an additional 10 minutes. The plate is then removed from theQIAVAC™ manifold and blotted dry on paper towels. The plate is thenre-attached to the QIAVAC™ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA is then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step is repeated with an additional60 μL water.

Example 13

[0174] Real-time Quantitative PCR Analysis of bcl-x mRNA Levels

[0175] Quantitation of bcl-x mRNA levels is determined by real-timequantitative PCR using the ABI PRISM™ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE or FAM, obtained from either Operon Technologies Inc.,Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ 7700 Sequence Detection System. In each assay,a series of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

[0176] PCR reagents are obtained from PE-Applied Biosystems, FosterCity, Calif. RT-PCR reactions are carried out by adding 25 μL PCRcocktail (1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTPand dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer,and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLpoly(A) mRNA solution. The RT reaction is carried out by incubation for30 minutes at 48□C. Following a 10 minute incubation at 95□ C. toactivate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol arecarried out: 95□C. for 15 seconds (denaturation) followed by 60□C. for1.5 minutes (annealing/extension). Bcl-x probes and primers are designedto hybridize to the human bcl-x nucleic acid sequence, using publishedsequence information (Boise et al., Cell, 1993, 74:597-608; GenBankaccession number L20121; locus name HSBCLXL), incorporated herein as SEQID NO: 1.

Example 14

[0177] Northern Blot Analysis of bcl-x mRNA Levels

[0178] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.).

[0179] Membranes were probed using QUICKHYB™ hybridization solution(Stratagene, La Jolla, Calif.) using manufacturer's recommendations forstringent conditions with an 822-base pair bcl-x specific probe preparedby PCR from bases 33-855 of human bcl-xl sequence (Boise et al., 1993,Cell 74:597-608; GenBank accession no. L20121). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA(Clontech, Palo Alto, Calif.). Hybridized membranes were visualized andquantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3(Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDHlevels in untreated controls.

Example 15

[0180] RNAse Protection Assay for Analysis of mRNA Levels

[0181] The ribonuclease (RNase) protection assay is a sensitive andspecific method for quantitating expression levels (Zinn, et al., Cell,1983, 34:865-79). The method is based on the hybridization of a targetRNA to an in vitro transcribed ³²P-labeled anti-sense RNA probe from aDNA template. RNase treatment follows, resulting in degradation ofsingle-stranded RNA and excess probe. The probe and target RNA areresolved by denaturing polyacrylamide gel electrophoresis with the“protected” probe visualized using autoradiography or beta imagingequipment. Template sets can be purchased (PharMingen Inc., San DiegoCalif.) which contain a series of biologically relevant templates, eachof distinct length and each representing a sequence in a distinct mRNAspecies. Each template set is capable of detecting up to 11 unique genemessages in a single reaction mix in addition to one or morehousekeeping genes, L32 and GAPDH, which serve as internal controls.These template sets allow for multiple determinations to be made from asingle sample. Multi-probe RPA can be performed on total RNApreparations derived by standard methods, without further purificationof poly-A+ RNA.

[0182] Oligonucleotides were evaluated for their respective effects onbcl-xs and bcl-xl mRNA levels along with total bcl-x mRNA levels, usingthe RIBOQUANT™ RNase protection kit (Pharmingen, San Diego Calif.). Allassays were performed according to manufacturer's protocols. Briefly,multi-probe DNA template sets were used to generate antisense RNAtranscripts radiolabeled with dUTP⁻³²P. The template set used forapoptosis genes was the human hAPO-2 set. These radiolabeled probes werehybridized overnight with typically 10 μg of total cellular RNA. Thereaction mixture was then digested with single-strand RNases to generatethe protected fragments which were electrophoresed through a 5%acrylamide/urea gel. Protected bands were visualized and quantitatedusing a PhosphorImager (Molecular Dynamics, Sunnyvale Calif.).

Example 16

[0183] Antisense Inhibition of bcl-x Expression—PhosphorothioateOligodeoxynucleotides

[0184] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of humanbcl-x RNA, using published sequences (Boise, L. H., et al., Cell, 1993,74, 597-608; Genbank Accession No. L20121, also listed as GenbankAccession No. Z23115; locus name “HSBCLXL,” incorporated herein as SEQID NO: 1). The oligonucleotides are shown in Table 1. Target sites areindicated by nucleotide numbers, as given in the sequence sourcereference (Genbank Accession No. L20121) to which the oligonucleotidebinds. All compounds in Table 1 are oligodeoxynucleotides withphosphorothioate backbones (internucleoside linkages) throughout. TABLE1 Human bcl-x Phosphorothioate Oligodeoxynucleotides SEQ ISIS NUCLEOTIDESEQUENCE¹ ID TARGET TARGET NO. (5′->3′) NO: SITE² REGION 11219CGGGTTCTCCTGGTGGCAAT 3 0907-0926 3′-UTR 11220 CAGTGTCTGGTCATTTCCGA 40827-0346 Stop 11221 AGCCCAGCAGAACCACGCCG 5 0797-0816 Coding, Exon 211222 GTTGAAGCGTTCCTGGCCCT 6 0748-0767 Coding, Exon 2 11223CAGTGCCCCGCCGAAGGAGA 7 0565-0584 Coding, Exon 1L³ 11224TCGCCTGCCTCCCTCAGCGC 8 0399-0418 Coding, Exon 1 11225CAGTGGCTCCATTCACCGCG 9 0323-0342 Coding, Exon 1 11226ATTCAGTCCCTTCTGGGGCC 10 0242-0261 Coding, Exon 1 11227AAAGTCAACCACCAGCTCCC 11 0151-0170 Coding, Exon 1 11228CCGGTTGCTCTGAGACATTT 12 0133-0152 AUG 11229 ACCAGTCCATTGTCCAAAAC 130093-0112 5′-UTR 11230 GAAGGGAGAGAAAGAGATTC 14 0001-0020 5′-UTR 11993TCATTCACTACCTGTTCAAA 15 0501-0520 Coding, Exon 1 12102AGCCCACCAGAAGGACCCCG 16 scrambled 11121 12103 CAGTGGCTCTCACCGCATCG 17scrambled 11225 12104 CAGCCCGCCTGCGAAGGAGA 18 scrambled 11223 12105AGCGCAGAACCACCACGCCG 19 scrambled 11221

[0185] Oligonucleotides were tested by Northern blot analysis asdescribed in Example 14. Oligonucleotides were tested in A549 cells at aconcentration of 400 nM. The results are Table 2: TABLE 2 Effect ofAntisense Phosphorothioate Oligodeoxynucleotides Targeted to Human Bcl-xon Bcl-x mRNA Levels ISIS SEQ NO. ID NO: TARGET REGION % CONTROL % INHIB11219 3 3′-UTR 6 94 11220 4 Stop 12 88 11221 5 Coding, Exon 2 11 8911222 6 Coding, Exon 2 35 65 11223 7 Coding, Exon 1L 9 91 11224 8Coding, Exon 1 6 94 11225 9 Coding, Exon 1 25 75 11226 10 Coding, Exon 134 66 11227 11 Coding, Exon 1 10 90 11228 12 AUG 31 69 11229 13 5′-UTR60 40 11230 14 5′-UTR 135 — 11993 15 Coding, Exon 1 142 —

[0186] SEQ ID NOs 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 inhibited bcl-xmRNA levels by greater than 50%. Of these, SEQ ID Nos 3, 4, 5, 7, 8 and11 inhibited bcl-x mRNA levels by greater than 85%.

Example 17

[0187] Dose Response Analysis of ISIS 11219 (SEQ ID NO: 3) and ISIS11224 (SEQ ID NO: 8)

[0188] Dose-response experiments were done to quantitate bcl-x mRNAlevels in A549 cells by Northern blot analysis after oligonucleotidetreatment with ISIS 11219 and 11224. The IC₅₀s obtained for thesecompounds were approximately 250 nM and 175 nM, respectively.

Example 18

[0189] Antisense Inhibition of bcl-x Expression—Phosphorothioate 2′-MOEGapmer Oligonucleotides

[0190] A second series of oligonucleotides targeted to human bcl-x wassynthesized. The oligonucleotide sequences are shown in Table 3. Targetsites are indicated by nucleotide numbers, as given in the sequencesource reference (Genbank Accession No. L20121), to which theoligonucleotide binds.

[0191] All compounds in Table 3 are chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings”. The wings arecomposed of 2′-O-methoxyethyl (2′-MOE) nucleotides. The internucleoside(backbone) linkages are phosphorothioate (P═S) throughout theoligonucleotide. Cytidine residues in the 2′-MOE wings are5-methylcytidines. TABLE 3 Nucleotide Sequences of Human Bcl-x ChimericOligonucleotides SEQ ISIS NUCLEOTIDE SEQUENCE¹ ID TARGET TARGET NO.(5′−>3′) NO: SITE² REGION 15998 TAATAGGGATGGGCTCAACC 20 0110-0129 5′-UTR15999 TCCCGGTTGCTCTGAGACAT 21 0135-0154 AUG 16000 GGGCCTCAGTCCTGTTCTCT22 0227-0246 Coding, Exon 1 16001 TCCATCTCCGATTCAGTCCC 23 0252-0271Coding, Exon 1 16002 AGGTGCCAGGATGGGTTGCC 24 0291-0310 Coding, Exon 116003 AGTGGCTCCATTCACCGCGG 25 0322-0341 Coding, Exon 1 16004CTTGCTTTACTGCTGCCATG 26 0380-0399 Coding, Exon 1 16005GCCGGTACCGCAGTTCAAAC 27 0422-0441 Coding, Exon 1 16006CTGTTCAAAGCTCTGATATG 28 0490-0509 Coding, Exon 1 16007TACCCCATCCCGGAAGAGTT 29 0520-0539 Coding, Exon 1L 16008AAAGGCCACAATGCGACCCC 30 0544-0563 Coding, Exon 1L 16009CTACGCTTTCCACGCACAGT 31 0581-0600 Coding, Exon 1L 16010TCCAAGCTGCGATCCGACTC 32 0623-0642 Coding, Exon 1L 16011CTGGATCCAAGGCTCTAGGT 33 0664-0683 Coding, Exon 1L 16012CCAGCCGCCGTTCTCCTGGA 34 0679-0698 Coding, Exon 1L 3′ end 16013TAGAGTTCCACAAAAGTATC 35 0699-0718 Coding, Exon 2 5′ end 16014AGCGTTCCTGGCCCTTTCGG 36 0743-0762 Coding, Exon 2 16015GTCATGCCCGTCAGGAACCA 37 0771-0790 Coding, Exon 2 16016TGAGCCCAGCAGAACCACGC 38 0799-0818 Coding, Exon 2 16017CAGTGTCTGGTCATTTCCGA 3 0827-0846 Stop 16018 GAGGGTAGAGTGGATGGTCA 390845-0864 3′-UTR 16019 GGAGGATGTGGTGGAGCAGA 40 0876-0895 3′-UTR 16020CGGGTTCTCCTGGTGGCAAT 2 0907-0926 3′-UTR

[0192] Oligonucleotides were tested by Northern blot analysis asdescribed in Example 14. Chimeric oligonucleotides were in A549 cells ata concentration of 200 nM. Results are shown in Table 4. Where present,“N.D.” indicates “not determined”. TABLE 4 Effect of Chimeric AntisenseOligonucleotides Targeted to Human Bcl-x on Bcl-x mRNA Levels SEQ ISISID NO. NO: TARGET REGION % CONTROL % INHIB 15998 20 5′-UTR 13 87 1599921 AUG 4 96 16000 22 Coding, Exon 1 4 96 16001 23 Coding, Exon 1 17 8316002 24 Coding, Exon 1 8 92 16003 25 Coding, Exon 1 12 88 16004 26Coding, Exon 1 5 95 16005 27 Coding, Exon 1 17 83 16006 28 Coding, Exon1 28 72 16007 29 Coding, Exon 1L 31 69 16008 30 Coding, Exon 1L N.D.N.D. 16009 31 Coding, Exon 1L 3 97 16010 32 Coding, Exon 1L 13 87 1601133 Coding, Exon 1L 31 69 16012 34 Coding, Exon 1L 3′ end 30 70 16013 35Coding, Exon 2 5′ end 85 15 16014 36 Coding, Exon 2 22 78 16015 37Coding, Exon 2 12 88 16016 38 Coding, Exon 2 28 72 16017 3 Stop 18 8216018 39 3′-UTR 40 60 16019 40 3′-UTR 40 60 16020 2 3′-UTR 20 80

[0193] Of the chimeric oligonucleotides tested, all but SEQ ID NO: 35inhibited bcl-x mRNA levels by at least 60%. Of these, SEQ ID NOs 20-27,31, 32, 37, 3 and 2 reduced bcl-x mRNA levels by 80% or more.

Example 19

[0194] Dose-Response Effect of ISIS 15999 and Mismatches on Bcl-x mRNALevels in A549 Cells

[0195] Dose-response experiments were done to quantitate bcl-x mRNAlevels in A549 cells by Northern blotting after oligonucleotidetreatment with ISIS 15999 and compounds based on the ISIS 15999sequences but with 2, 4, 6 or 8 mismatches from the 15999 sequence. TheIC₅₀ obtained for ISIS 15999 was estimated to be well below 25 nMbecause the lowest oligonucleotide dose tested, 25 nM, gaveapproximately 70% reduction of bcl-x mRNA levels and oligonucleotidedoses of 50 nM to 200 nM gave inhibition of greater than 90%, withnearly complete ablation of bcl-x mRNA at the highest dose.Oligonucleotides with 2 or 4 mismatches had IC₅₀s of approximately 200nM, the highest dose tested, and oligonucleotides with 6 or 8 mismatchesdid not inhibit mRNA levels below control levels.

Example 20

[0196] Dose-Response Effect of ISIS 16009 and Mismatches on Bcl-x mRNALevels in A549 Cells

[0197] Dose-response experiments were done to quantitate bcl-x mRNAlevels in A549 cells by Northern blot analysis after oligonucleotidetreatment with ISIS 16009 and compounds based on the ISIS 16009 sequencebut with 2, 4, 6 or 8 mismatches from the 16009 sequence. The IC₅₀obtained for ISIS 16009 was estimated to be 40-50 nM. IC₅₀s could not beobtained for mismatched oligonucleotides because 50% inhibition of mRNAlevels was not achieved at any of the doses tested (25-200 nM).

Example 21

[0198] Optimization of ISIS 15999 and 16009

[0199] Several analogs of ISIS 15999 (SEQ ID NO: 21) and ISIS 16009 (SEQID NO: 31) were prepared. These had various placements of2′-O-methoxyethyl (2′-MOE) modifications and uniformly phosphorothioate(P═S) backbones or chimeric backbones in which the 2′-O-methoxyethylwings had phosphodiester (P═O) backbones and the deoxy gap had aphosphorothioate (P═S) backbone. These compounds are shown in Table 5.All 2′-MOE cytosines were 5-methyl-cytosines (5meC). TABLE 5 Analogs ofISIS 15999 and 16009 SEQ ISIS ID No. SEQUENCE¹ NO: 15999TsCsCsCsGsGsTsTsGsCsTsCsTsGsAsGsAsCsAsT 21 17791ToCoCoCoGsGsTsTsGsCsTsCsTsGsAsGoAoCoAoT 21 17958TsCsCsCsGsGsTsTsGsCsTsCsTsGsAsGsAsCsAsT 21 17959TsCsCsCsGsGsTsTsGsCsTsCsTsGsAsGsAsCsAsT 21 16009CsTsAsCsGsCsTsTsTsCsCsAsCsGsCsAsCsAsGsT 31 17792CoToAoCoGsCsTsTsTsCsCsAsCsGsCsAoCoAoGoT 31 17956CsTsAsCsGsCsTsTsTsCsCsAsCsGsCsAsCsAsGsT 31 17957CsTsAsCsGsCsTsTsTsCsCsAsCsGsCsAsCsAsGsT 31 17619CsTsAsCsGsCsTsTsTsCsCsAsCsGsCsAsCsAsGsT 31 ¹Emboldened residues are2′-MOE residues (others are 2′-deoxy). All 2′-MOE cytosines were5-methyl-cytosines; linkages are indicated as “s” for phosphorothioate(P = S) linkages and “o” for phosphodiester (P = O) linkages.

Example 22

[0200] Dose-Response Effect of ISIS 16009 and Analogs on Bcl-x mRNALevels in A549 Cells

[0201] Dose-response experiments were done to quantitate bcl-x mRNAlevels in A549 cells by Northern blot analysis after oligonucleotidetreatment with ISIS 16009 and analogs shown in Table 5. Oligonucleotideswere tested at concentrations of 50, 100, 150 and 200 nM. IC₅₀s obtainedare shown in Table 6. An IC₅₀ was not obtained for ISIS 17619 (P═S, fulldeoxy) because 50% reduction in bcl-x mRNA was not achieved at doses upto 200 nM. TABLE 6 IC₅₀s for analogs of SEQ ID NO: 31 ISIS IC₅₀ No.SEQUENCE (nM) 16009 CsTsAsCsGsCsTsTsTsCsCsAsCsGsCsAsCsAsGsT 35 17792CoToAoCoGs CsTsTsTsCsCsAsCsGsCsAoCoAoGoT 143 17956CsTsAsCsGsCsTsTsTsCsCsAsCsGsCsAsCsAsGsT 47 17957CsTsAsCsGsCsTsTsTsCsCsAsCsGsCsAsCsAsGsT 43 17619CsTsAsCsGsCsTsTsTsCsCsAsCsGsCsAsCsAsGsT >200

Example 23

[0202] Western Blot Analysis of bcl-x Protein Levels

[0203] Western blot analysis (immunoblot analysis) was carried out usingstandard methods. Generally, cells were harvested 16-20 hours afteroligonucleotide treatment, washed once with PBS, suspended in Laemmlibuffer (100 μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGEgel. Gels were run for 1.5 hours at 150 V, and transferred to membranefor western blotting. Appropriate primary antibody directed to bcl-x wasused, with a radiolabelled or fluorescently labeled secondary antibodydirected against the primary antibody species. Bands were visualizedusing a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

[0204] ISIS 15999 and 16009 were tested for the ability to reduce bcl-xprotein levels in A549. Both compounds were found to reduce bcl-xprotein levels in a dose-dependent manner.

Example 24

[0205] Effect of ISIS 15999 on bcl-x Protein Levels in SEM-K2 Cells

[0206] SEM-K2 is a human cell line derived from a patient suffering froma t(4;11) acute lymphoblastic leukemia. Pocock, C. F. E. et al., Br. J.Haematol. (1995), 90(4), 855-67. SEM-K2 cells in exponential phase ofgrowth were maintained in RPMI 1460 medium (Life Technologies, Inc.,Gaithersburg, Md.) supplemented with 10% fetal bovine serum, 2 mMglutamine and penicillin/streptomycin, at 37□C. in 5%CO2/95% air. Cellswere transferred in 1 mL volumes at approximately 1-5×10⁶ cells/ml to24-well plates. After one hour, 10 μM of ISIS 15999 or scrambled control15691 (GACATCCCTTTCCCCCTCGG; SEQ ID No. 41) was added to wells, withoutcationic lipid. At 24 hours and 48 hours repeat doses of 5 μMoligonucleotide were added. Cells were analyzed at 72 hours.

[0207] For Western blot and flow cytometric protein analysis, cells werewashed once in phosphate-buffered saline and pelleted by centrifugationat 1200 rpm for 5 minutes, and resuspended in cell lysis buffer (5MNaCl, 0.1 M HEPES, 500 mM sucrose, 0.5M EDTA, 100 mM spermine, 1 mg/mlaprotinin, 10% Triton X-100) for 15 minutes on ice. Total protein wasquantified spectrophotometrically (BioRad) and 100 μg lysate was loadedonto 15% polyacrylamide gels and run at 200V for 45 minutes. Followingprotein transfer to nitrocellulose membrane, blots were immunostainedwith mouse monoclonal bcl-x antibody (Transduction Laboratories,Lexington Ky.) followed by horseradish peroxidase-conjugated goatanti-mouse secondary antibody (Santa Cruz Biotechnology, Inc., SantaCruz Calif.). Protein was qualitatively visualized by ECL (Amersham,Piscataway N.J.) and exposure on photographic film. Cells for flowcytometry were permeabilized by fixation on ice in 70% ethanol for 30minutes. Two-step immunostaining employed bcl-xl antibody (JacksonImmunoResearch Lab., Westr Grove Pa.) followed by fluoresceinisothiocyanate (FITC)-conjugated anti-mouse antibody. Cells wereanalyzed using a FACScalibur™ running Cellquest software (BectonDickinson, Franklin Lakes, N.J.). A lymphoid-enriched gate was used foracquisition of 50,000 events. Fluorescence detector 1 was used withlogarithmic amplification for detection of FITC fluorescence.

[0208] Median fluorescence intensity of bcl-x-stained SEM-K2 cells wasmeasured using WINMIDI 2.5 software. SEM-K2 express bcl-x at relativelyhigh levels as detected by immunofluorescence. Median fluorescenceintensity for the population treated with 10 μM ISIS 15999 was comparedwith positive and negative control samples, respectively, and thepercentage reduction in bcl-x expression was calculated. Using 10 μMISIS 15999, 50% reduction in bcl-x was measured at 48 hours afterinitial treatment.

Example 25

[0209] SCID-Human Leukemia Model and in Vivo bcl-xl Antisense Treatment

[0210] 10⁷ SEM-K2 cells in exponential phase of growth were injectedsubcutaneously into 8 SCID-NOD mice as a bolus (suspended in sterilesaline). Engraftment and tumor formation occurred over a 2-3 weekperiod. Micro Alzet pumps (Alza, Newark Del.) capable of delivering acontinuous subcutaneous infusion over 14 days were used to deliver adose of 100 μg per day (equivalent to 5 mg/kg) of ISIS 16009 andscrambled control ISIS 15691 into two groups of three animals,respectively. The remaining two animals received vehicle (sterilesaline) only.

[0211] The expression of bcl-x measured in SEM-K2 cells from SCID-huxenografts was shown to be dramatically reduced by 14-day infusion of 5mg/kg/day equivalent (100 μg/day) of ISIS 16009. Mean reductions inexpression of approximately 90% (n=3) compared to control (p<0.01) weremeasured using quantitative flow cytometry. This was compared tostatistically insignificant (<20%, p<0.3) reductions in bcl-x expressionby scrambled control oligonucleotide ISIS 15691 (n=3).

Example 26

[0212] Stimulation and Measurement of Apoptosis

[0213] Xenografts were removed after sacrifice and mechanicallydispersed into large volumes of medium. Leukocytes were purified bydensity gradient centrifugation and washed with medium beforeresuspending in 1 ml volumes at 1-5×10⁶ cells/ml. Cells were incubatedat 37□ C. in 95% humidified air/5% CO₂ for 2 hours prior to induction ofapoptosis with 20 μg/ml VP-16 (Etoposide) over 24 hours. Each xenograftcell suspension treated with VP-16 was paired with a respective negativecontrol. Apoptosis was assessed nonspecifically using quantification oflight scatter changes; reduction in side scatter (due to chromatincondensation) and reduction in forward scatter (due to cell shrinkage)are early changes associated with apoptosis. Bimodal populationdistributions consisting of apoptotic and non-apoptotic cells could bemeasured respectively allowing estimation of an apoptotic index fortreated and negative control. Fold increase in apoptosis was calculatedfrom their ratio. More specific determination of apoptosis was achievedusing the Apo-Alert Caspase-3 Colorimetric Assay Kit (Clontech, PaloAlto Calif.). This is a DEVD-specific caspase assay, a quantitativeassay for the activity of caspase-3, a member of the caspase familythought to mediate apoptosis in most mammalian cell types. This assayutilizes a synthetic tetrapeptide, Asp-Glu-Val-Asp (DEVD; SEQ ID No.42), labeled with either a fluorescent mol., 7-amino-4-trifluoromethylcoumarin (AFC), or a calorimetric mol., p-nitroanilide (pNA) assubstrates. DEVD-dependent protease activity is assessed by detection ofthe free AFC or pNA cleaved from the substrates. Cell lysates wereincubated with DEVD conjugated to paranitroanilide, a calorimetricsubstrate cleaved by CPP32 (caspase-3) and detectable using calorimetricspectrophotometry at 405 nm. The fold increase in OD_(405nm) was used todetermine the net VP-16-induced apoptosis.

[0214] Sequence specific increases in CPP32 activation measured byDEVD-paranitroanilide cleavage were detected. A 46% increase inOD_(405nm) was detected above control xenografts for ISIS 16009-treatedSCID-hu models (n=3). Scrambled control oligonucleotide-treatedxenografts yielded very little change in VP-16-induced apoptosis (n=3)which was quantitively similar to that of xenografts not treated witholigonucleotide. A scatter plot of bcl-x expression versus fold increasein CPP32 activity (pooled from all 8 animals) revealed a positivecorrelation (r_(s)>8) suggesting a definite relationship between bcl-xexpression and apoptosis sensitivity to VP-16. A similar profile ofchange was detected in light scatter measurements of apoptosis.Sequence-specific increase in apoptotic index was associated with ISIS16009; this was not seen in the ISIS 15691(control) treated group northe untreated control group. Again, a pooled scatter plot shows apositive correlation (r_(s)>8) for bcl-x versus apoptosis (lightscatter), suggesting a strong relationship.

Example 27

[0215] Measurements of Cell Viability and Clonogenicity

[0216] Propidium iodide (20 μg/ml) was added to PBS-washed cells andflow cytometry was performed using fluorescence detector 3 vs. sidescatter. This charged dye is excluded from live cells and may be used todetect dead or late apoptotic cells in which propidium iodide readilybecomes incorporated. Viable and non-viable cells were counted and aviability fraction was computed. The ratio of VP-16 to negative controlviability was determined to provide a measure of VP-16-induced reductionin viability. Viable cells metabolize (reduce) the tetrazolium salt MTT(Roche Molecular Biochemicals, Indianapolis Ind.) to a purple formozanproduct which is detectable by spectophotometry, thus providing a meansfor quantitation. The fold decrease in cell viability was determined at96 hours. Cell proliferation increases MTT metabolizing capacity invitro, thus providing an index of clonogenic growth. The fold reductionin VP-16-treated versus negative control cells was determined to providea measure of net cytotoxic effect on clonogenicity.

[0217] A reduction in MTT viability was observed and shown to besequence-specific. A pooled scatter plot (n=8) of bcl-x expressionversus fold decrease in metabolic viability shows a negative correlation(r_(s)=−5) is seen, which is consistent with a role of bcl-x inproviding a survival advantage in cells stimulated to undergo apoptosisby VP-16.

Example 28

[0218] Effect of Antisense Oligonucleotides on Expression of bcl-xs andbcl-xl Transcripts

[0219] Additional oligonucleotides were designed to target particularareas of exon 1 and exon 2 of human bcl-x, particularly around the exon1/exon 2 splice site and in sequence regions present in bcl-xl but notin bcl-xs. These oligonucleotides are shown in Table 7. All backbonelinkages are phosphorothioates; All 2′ MOE cytosines are5-methylcytosines. TABLE 7 Oligonucleotides targeted to exon1/exon 2 ofhuman bcl-x SEQ Target ID ISIS # Sequence Target Region site NO: 16009CTACGCTTTCCACGCAC Coding, Exon 581-600 31 AGT 1L 16968 CTCCGATGTCCCCTCAA6 base 16009 43 AGT mismatch 15999 TCCCGGTTGCTCTGAGA AUG 135-154 21 CAT16972 TCACGTTGGCGCTTAGC 6 base 15999 44 CAT mismatch 16011CTGGATCCAAGGCTCTA Coding, Exon 664-683 33 GGT 1L 22783 CTGGATCCAAGGCTCTACoding, Exon 664-683 33 GGT 1L 16012 CCAGCCGCCGTTCTCCT Coding, Exon679-698 34 GGA 1L 3′ end 22784 CCAGCCGCCGTTCTCCT Coding, Exon 679-698 34GGA 1L 3′ end 16013 TAGAGTTCCACAAAAGT Coding, Exon 2 699-718 35 ATC5′ end 22781 TAGAGTTCCACAAAAGT Coding, Exon 2 699-718 35 ATC 5′ end22782 CAAAAGTATCCCAGCCG Coding, Exon 689-708 45 CCG ½ splice 22785GCCGCCGTTCTCCTGGA Coding, Exon 676-695 46 TCC 1L 23172 GTTCCTGGCCCTTTCGGCoding, Exon 2 740-759 47 CTC 23173 CAGGAACCAGCGGTTGA Coding, Exon 2760-779 48 AGC 23174 CCGGCCACAGTCATGCC Coding, Exon 2 780-799 49 CGT23175 TGTAGCCCAGCAGAACC Coding, Exon 2 800-819 50 ACG

[0220] Human bcl-x has two forms, a long form known as bcl-xl and ashort form known as bcl-xs. These result from alternatively spliced mRNAtranscripts. The protein of bcl-xl is similar in size and structure tobcl-2, and could inhibit cell death upon growth factor withdrawal. Theprotein of bcl-xs is 63 amino acids shorter than bcl-xl. It couldinhibit the bcl-2 function, thus promoting programmed cell death(apoptosis). Oligonucleotides were evaluated for their respectiveeffects on bcl-xs and bcl-xl mRNA levels along with total bcl-x mRNAlevels, using the RIBOQUANT™ RNase protection kit (Pharmingen, San DiegoCalif.). All assays were performed according to manufacturer'sprotocols. Results are shown in Table 8. TABLE 8 Effect of antisenseoligonucleotides on bcl-xs and bcl-xl % CONTROL bcl-xs/ SEQ ID % CONTROL% CONTROL total bcl-xl ISIS # NO bcl-xs bcl-xl bcl-x (%) bcl-xs/bcl-xl*no — 100 100 100 17.56 1 oligo 16009 31 20 24 24 12.45 0.71 16968 43 2015 21 20.18 1.15 15999 21 ND** ND ND — — 16972 44 60 91 87 11.68 0.6716011 33 ND ND ND — — 22783 33 620 35 120 293.10 16.69 16012 34 48 63 6113.17 0.75 22784 34 204 72 92 48.63 2.77 16013 35 60 83 82 12.46 0.7122781 35 ND ND ND — — 22782 45 64 76 75 15.72 0.89 22785 46 248 53 8380.14 4.56 23172 47 84 77 79 19.38 1.10 23173 48 ND ND ND — — 23174 4956 67 66 14.93 0.85 23175 50 52 82 78 11.44 0.65

[0221] ISIS 22783, a fully 2′-MOE, fully-phosphorothioateoligonucleotide targeted to exon 1 of the bcl-xl transcript (not thebcl-xs transcript), is able to change the ratio of bcl-xs to bcl-xl from17% to 293%, without reducing the total bcl-x mRNA level in A549 cells.That is, it reduced the bcl-xl form but dramatically increased thebcl-xs form.

[0222] ISIS 22783 was tested by RNAse protection assay for ability toinhibit bax, another apoptotic gene. It had no effect on bax mRNAlevels. ISIS 22783 is also fully complementary to the murine bcl-x mRNAwhich makes it useful for animal studies.

Example 29

[0223] Antisense Redirection of bcl-x Splice Products in Other CellLines

[0224] In addition to its activity in A549 cells shown in the previousexamples, ISIS 22783 was able to similarly alter the bcl-xl/xs spliceproduct ratio in human 293T embryonic kidney carcinoma cells, humanC8161 melanoma cells and HeLa cells.

Example 30

[0225] Additional Modifications of the ISIS 22783 Sequence

[0226] It is believed that modifications which provide tight binding ofthe antisense compound to the target and resistance to nucleases arealso particularly useful in targeting splice sites. One suchmodification is the 2′-methoxyethoxy (2′-MOE) modification. Otherexamples of such modifications include but are not limited to sugarmodifications including 2′-dimethylaminooxyethoxy (2′-DMAOE) and2′-acetamides; backbone modifications such as morpholino, MMI and PNAbackbones, and base modifications such as C-5 propyne.

[0227] An antisense compound which has the ISIS 22783 sequence and a2′-DMAOE modification on each sugar was compared to its 2′-MOE analogfor ability to alter the ratio of bcl-x splice products. The results areshown in Table 9. TABLE 9 Comparison of the 2′-MOE and 2′-DMAOE analogsof the ISIS 22783 sequence for effect on bcl-xs/bcl-xl ratio SEQ IDOligo Ratio (approx.) of Chemistry NO: Concentration bcl-xs/bcl-xl2′-MOE 33 100 4.5 ″ 200 8.5 ″ 400 18 2′-DMAOE ″ 100 1.8 ″ 200 4 ″ 400 12

[0228] Thus compared to the 2′-MOE compound, the 2′-DMAOE compoundshowed qualitatively similar, though quantitatively slightly less,ability to alter the ratio of bcl-xs to bcl-xl splice products. 2′-DMAOEcompounds are therefore preferred.

[0229] Preliminary experiments with a morpholino-backbone compound withthe 22783 sequence showed good activity as measured by RPA. Compoundswere prepared according to U.S. Pat. No. 5,034,506, and transfected intoHeLa cells by scrape-loading. Summerton et al., 1997, Antisense NucleicAcid Drug Dev., 7,63-70.

[0230] A peptide-nucleic acid (PNA) oligonucleotide having the ISIS22783 sequence (SEQ ID NO: 33) was synthesized as described in Example 4hereinabove. The oligonucleotide (ISIS 32262) was uniformly modifiedwith a PNA backbone throughout. A 5-base mismatch compound (ISIS 32263,SEQ ID NO: 52) was also synthesized as a full PNA. HeLa cells weretransfected with these compounds by electroporation with a 200V pulseusing a BTX Electro Cell Manipulator 600 (Genetronics, San Diego Calif.)and RNA was isolated 24 hours later. The effects of the PNA compounds onbcl-x splicing is shown in Table 10. TABLE 10 Effect of PNA analogs ofthe ISIS 22783 sequence on bcl-xs/bcl-xl transcript ratio SEQ ID OligoRatio (approx.) of Chemistry NO: Concentration bcl-xs/bcl-xl No oligo1.0 PNA 33 1 μM 1.5 ″ 2 μM 2.0 ″ 5 μM 3.5 ″ 10 μM  6.5 ″ 15 μM  7.75 ″25 μM  11.5 PNA 52 1 μM 1.5 (control) ″ 2 μM 0.75 5 μM 0.75 10 μM  1.015 μM  0.75 25 μM  1.0

[0231] Thus the PNA compound is also able to alter bcl-x splice productsand is therefore preferred.

Example 31

[0232] Antisense Inhibition of bcl-xl RNA and Protein in PrimaryKeratinocyte (Skin) Cells

[0233] Normal human neonatal keratinocytes (hKn) cells (CascadeBiologics, Inc., Portland, Oreg.), which are primary keratinocyte cells,were treated with ISIS 16009 and bcl-xl mRNA and protein levels wereanalyzed. Cells were grown in 100 mm tissue culture dishes until 70-70%confluent. Cells were transfected with oligonucleotide in the presenceof cationic lipid as follows. Lipofectin™ (Gibco/BRL) was used at aconcentration of 10 μg/ml Opti-MEM™ (Gibco/BRL). A 5 ml mixture ofOpti-MEM™, Lipofectin™ and varying amounts of oligonucleotide wasequilibrated for 30 minutes at room temperature. The cells were washedtwice with Opti-MEM™ and then treated with theOpti-MEM™/Lipofectin™/oligonucleotide mixture for 4 hours. The mixturewas then replaced with normal growth medium. Cells were harvested oranalyzed 24 hours later at the indicated times. Antisense or scrambledoligonucleotides were transfected at concentrations of 50, 100, 200 or300 nM. Total RNA was harvested from cells using the RNAeasy™ method(Qiagen, Valencia Calif.). Equal amoutns of RNA (10-20 μg) were resolvedin 1.2% agarose gels containing 1.1% formaldehyde and transferredovernight to nylon membranes. Membranes were probed with ³²P-labeledbcl-xl, bcl-2 or G3PDH cDNAs. Radioactive probes were generated usingthe Strip-EZ™ kit (Ambion, Austin, Tex.) and hybridized to the membraneusing QuikHyb™ solution (Stratagene, La Jolla Calif.). The amount of RNAwas quantified and normalized to G3PDH mRNA levels using a MolecularDynamics PhosphorImager.

[0234] ISIS 15999 and 16009 showed comparable (>90%) inhibition ofbcl-xl mRNA expression at a 200 nM screening dose. ISIS 16009 was foundto decrease the bcl-xl mRNA in hKn cells in a concentration-dependentmanner with an IC50 of approximately 50 nM. The scrambled controloligonucleotide for ISIS 16009, ISIS 20292 (CGACACGTACCTCTCGCATT;bold=2′-MOE, remainder is 2′-deoxy; backbone is phosphorothioate; SEQ IDNO:51) had no effect. The RNA expression of other apoptotic genes [A1,Bad, Bak, bcl-2, as detected by RNAse protection assay using theRiboQuant™ human Apo-2 probe set and protocol (Pharmingen, San DiegoCalif.)] or G3PDH (as detected by Northern blot analysis) was unaffectedby ISIS 16009 or 15999.

[0235] It should be noted that while ISIS 16009 and 15999 hybridize toregions of the bcl-x gene that are present in both bcl-xl and bcl-xs,there is no significant expression of bcl-xs in HUVEC, hKn or A549cells, so these compounds are considered as bcl-xl inhibitors because ofthe insignificant contribution of bcl-xs to the effects demonstrated.

[0236] The level of bcl-xl protein in antisense-treated hKn cells wasexamined by Western analysis. Whole cell extracts were prepared bylysing the cells in RIPA buffer (1×PBS, 1% NP40, 0.1% deoxycholate, 0.1%SDS, containing the complete protease inhibitor mix (BoehringerMannheim). Protein concentration of the cell extracts was measured byBradford assay using the BioRad kit (BioRad, Hercules, Calif.). Equalamounts of protein (10-50 μg) were then resolved on a 10% SDS-PAGE gel(Novex, San Diego Calif.) and transferred to PVDF membranes (Novex). Themembranes were blocked for one hour in PBS containing 0.1% Tween-20 and5% milk powder. After incubation at room temperature with a 1:500dilution of a mouse monoclonal bcl-x antibody (Transduction Labs,Lexington Ky.), the membranes were washed in PBS containing 0.1%Tween-20 and incubated with a 1:5000 dilution of goat anti-mousehorseradish peroxidase conjugated antibody in blocking buffer. Membraneswere washed and developed using Enhanced Chemiluminescent (ECL)detection system (Amersham, Piscataway N.J.).

[0237] 24 hours after treatment, bcl-xl protein levels decreased in aconcentration-dependent manner. Treatment resulted in greater than 70%inhibition of bcl-xl protein. This decrease remained low for over 48hours after transfection.

Example 32

[0238] Ultraviolet Irradiation of A549 and hKn Cells

[0239] A549 and hKn cells were irradiated with UV-B light when they wereapproximately 70-80% confluent or 24 hours after treatment witholigonucleotide. Immediately before UV-B treatment, cells were washedtwice with PBS and then exposed to UV-B light in a Stratalinker UVCrosslinker 1800 model (Stratagene, La Jolla Calif.) containing 515-watt 312 nm bulbs. The cells were exposed to 50 mJ/m², 100 mJ/m² or200 mJ/m² of UV-B radiation. The dose of UV-B radiation was calibratedusing a UVX radiometer (UVP). Following UV irradiation, the cells wereincubated in the standard medium for an additional 24 hours. Controlplates for UV-B treatment were simply washed 3 times in PBS andincubated in the standard medium for an additional 24 hours. Cells wereexamined for apoptosis by staining the ethanol-fixed cell nuclei withpropidium iodide and examining the DNA content by flow cytometry.Apoptotic cells were identified by their sub-diploid DNA content. Cellswere washed twice with cold PBS and resuspended in 1 ml of 70% ethanol.After 1 hour incubation at room temperature, cells were washed in PBSand resuspended in 1 ml propidium iodide staining solution (50 μg/mlpropidium iodide, 0.5 U/ml RNAse A, 2000 U/ml RNase T1. After 30 minutesat room temperature, cell cycle analysis was performed by flow cytometryusing a Becton Dickinson Calibur FACS analyzer. The fluorescence ofindividual nuclei of 10,000 cells was measured using a FACScan flowcytometer. Results were expressed as percentage apoptotic cells.

Example 33

[0240] Antisense Sensitization of A549 Cells to UV-induced Cell Death

[0241] A549 cells were treated with 100 nM ISIS 22783 or the 5-mismatchISIS 26080 (CTGGTTACACGACTCCAGGT; SEQ ID NO: 52) and exposed toultraviolet (UV) radiation. The percent apoptotic cells was quantitatedby propidium iodide staining according to standard methods. Results areshown in Table 11. TABLE 11 Combination of ISIS 22783 and UV irradiation% Apoptotic Compound UV mJ/M² cells (approx) SEQ ID NO: No oligo 0 <1 501 100 10 200 22 ISIS 22783 0 2 33 50 4 ″ 100 33 ″ 200 27 ″ ISIS 26080 01 52 50 6 ″ 100 15 ″ 200 29 ″

[0242] Thus the response to the apoptotic stimulus (irradiation) hasbeen changed after antisense treatment resulting in increased apoptosis.

Example 34

[0243] Antisense Sensitization of Primary Keratinocyte Cells toUV-Induced Cell Death

[0244] Exposure of skin to UV radiation and other DNA-damaging agentstriggers a protective response against DNA damage. We examined the roleof bcl-xl in resistance of keratinocytes to UV-B-induced apoptosis. Asfor A549 cells, treatment of hKn cells with ISIS 16009 sensitized thecells to apoptosis induced by UV-B irradiation. Less than 7% of cellstreated with no oligonucleotide or control oligonucleotide andirradiated with 100 mJ/m² UV-B irradiation underwent apoptosis. Incontrast, when the cells were transfected with 300 nM of the bcl-xlinhibitor ISIS 16009 and treated with the same dose of UV radiation,over 35% of the cells became apoptotic.

Example 35

[0245] Cisplatinum Treatment of A549 and hKn Cells

[0246] Cisplatinum is an alkylating agent that causes DNA damage and caninduce apoptosis. Gill and Windebank, 1998, J. Clin. Invest.101:2842-2850. A549 and hKn cells were treated with cisplatinum whenthey were approximately 70-80% confluent or 24 hours after treatmentwith oligonucleotide. Cis-diamminedichloroplatinum II (Cisplatinum,Sigma, St. Louis Mo.) was dissolved in distilled water at aconcentration of 1 mg/ml, and was added to the standard medium at a doserange of 0.5 to 10 μg/ml and incubated with cells for 24 hours.

Example 36

[0247] Antisense Sensitization of A549 Cells to Cisplatinum-Induced CellDeath

[0248] A549 cells were treated with 100 nM ISIS 22783 or the 5-mismatchISIS 26080 and cisplatinum at various doses. The percent apoptotic cellswas quantitated by propidium iodide staining according to standardmethods. Results are shown in Table 12. TABLE 12 Combination of ISIS22783 and Cisplatinum Cisplatinum % Apoptotic Compound dose (μg/ml cells(approx) SEQ ID NO: No oligo 0 4 1 5 10 8 50 18 ISIS 22783 0 3 33 1 6 ″10 13 ″ 50 27 ″ ISIS 26080 0 3 52 1 2 ″ 10 7 ″ 50 21 ″

[0249] Thus the cells have been sensitized to the apoptotic stimulus (inthis case a cytotoxic chemotherapeutic drug) after antisense treatmentresulting in increased apoptosis.

Example 37

[0250] Antisense Sensitization of hKn Cells to Cisplatinum-Induced CellDeath

[0251] When hKn cells transfected with no oligonucleotide or the controloligonucleotide ISIS 26080 were treated with 0.5 μg/ml cisplatinum, lessthan 10% of the cells became apoptotic. Treatment of hKn cells with thecombination of the bcl-xl inhibitor ISIS 16009 and the same dose ofcisplatinum caused over 25% of the cells to die.

Example 38

[0252] Antisense Sensitization of A549 Cells to Taxol-Induced Cell Death

[0253] A549 cells were treated with 100 nM ISIS 22783 or the 5-mismatchISIS 26080 and taxol at various doses. The percent apoptotic cells wasquantitated by propidium iodide staining according to standard methods.Results are shown in Table 13. TABLE 13 Combination of ISIS 22783 andTaxol Taxol dose % Apoptotic Compound (μg/ml cells (approx) SEQ ID NO:No oligo 0 2 5 3 10 7 30 16 ISIS 22783 0 8 33 5 8 ″ 10 15 ″ 30 26 ″ ISIS26080 0 2 52 5 3 ″ 10 10 ″ 30 15 ″

[0254] Thus the response to the apoptotic stimulus (here a cytotoxicchemotherapeutic drug) has been changed after antisense treatmentresulting in increased apoptosis.

Example 39

[0255] Treatment of Human Umbilical Vein Endothelial Cells (HUVEC) withAntisense Oligonucleotide to bcl-x and/or Apoptotic Stimuli

[0256] Human umbilical vein endothelial cells (HUVEC) were obtained fromClonetics (San Diego Calif.) and cultivated in endothelial growth medium(EGM) supplemented with 10% fetal bovine serum. Cells were used betweenpassages 2 and 5 and were used at approximately 80% confluency. Cellswere washed three times with pre-warmed (37° C.) Opti-MEM™.Oligonucleotides were premixed with 10 μg/ml Lipofectin™ in Opto-MEM™ atan oligonucleotide concentration of 50 nM ISIS 16009. Cells wereincubated with oligonucleotide for 4 hours at 37° C. after which themedium was removed and replaced with standard growth medium. Treatmentwith C6-ceramide (Calbiochem, San Diego Calif.), staurosporine(Calbiochem), or z-VAD.fmk (Calbiochem), if any, was done 24 hours afteroligonucleotide treatment. Bcl-xl mRNA levels were measured by Northernblot analysis and found to be decreased to approximately 5% of control,with an apparent IC₅₀ of less than 20 nM. Bcl-xl protein levels weremeasured by Western analysis and found be approximately 5% of control.Apoptotic cells with fragmented DNA were identified by flow cytometryanalysis of hypodiploid cells. Inhibition of bcl-x protein (which isvirtually all bcl-xl in these cells) caused 10-25% of the cellpopulation to undergo apoptosis.

Example 40

[0257] Sensitization of HUVEC Endothelial Cells to Apoptotic Stimuli bythe bcl-xl Inhibitor ISIS 16009

[0258] Staurosporine (a protein kinase inhibitor) and C6-ceramide (alipid second messenger) have been shown to induce apoptosis in many celltypes. Treatment of HUVEC with low doses of staurosporine (2 nM) orC6-ceramide (5 μM) alone did not cause a significant increase incellular DNA fragmentation (a measure of apoptosis) over the backgroundof 10-25% apoptotic cells. However, cells treated with ISIS 16009 toreduce levels of bcl-x were more sensitive to these doses of apoptoticstimuli, with over 50% apoptotic cells in samples treated with ISIS16009 and staurosporine, and over 40% apoptosis in cells treated withISIS 16009 and ceramide. Thus inhibition of bcl-x sensitizes cells tothese apoptotic stimuli. The apoptosis caused by bcl-x inhibition, orbcl-x plus staurosporine or ceramide, was prevented by treatment ofcells with the caspase inhibitors z-VAD.fmk or z-DEVD.fmk (Calbiochem,San Diego Calif.).

Example 41

[0259] Measurement of Mitochondrial Dysfunction

[0260] To evaluate the mitochondrial transmembrane potential (usuallyabbreviated as □m, cells were incubated with the cationic lipophilic dyeMitoTracker Orange CMTMRos (Molecular Probes, Eugene Oreg.) at aconcentration of 150 nM for 15 minutes at 37° C. in the dark. Controlcells were simultaneously treated with 50 μM of the protonophores,carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Calbiochem, San DiegoCalif.)which disrupts mitochondrial transmembrane potential. Bothadherent and floating cells were collected, washed once with 1× PBS/2%BSA, and fixed in 1 ml PBS containing 4% paraformaldehyde for 15 minutesat room temperature while shaking. Fixed cells were stored in the darkat 4° C. for 1 day prior to analysis by flow cytometry.

Example 42

[0261] Effect of bcl-x Inhibition on Mitochondrial Integrity

[0262] One proposed mechanism by which cells are protected fromapoptosis is by protection of mitochondrial function. Treatment of HUVECwith the bcl-x inhibitor ISIS 16009 resulted in a reduction inmitochondrial transmembrane potential, which was potentiated by eitherstaurosporine or ceramide. The loss of mitochondrial integrity caused byeither bcl-x antisense inhibitor alone or antisense plus staurosporine(but not antisense plus ceramide) was prevented by treatment of HUVECwith the caspase inhibitor z-VAD.fmk (Calbiochem, San Diego Calif.).

Example 43

[0263] Additional 2′-MOE Oligonucleotides Designed to Alter Splicing ofHuman bcl-xl

[0264] An additional series of uniform 2′-MOE oligonucleotides weredesigned to target the region upstream from or overlapping the 5′ splicesite at nucleotide 699 of human bcl-xl. The purpose of this was tooptimize the effect on splice products, increasing the ratio ofbcl-xs/bcl-xl transcripts produced. The oligonucleotides are shown inTable 14. Backbones are phosphorothioate throughout. All nucleotidenumbers correspond to those on Genbank accession no. Z23115 except forISIS 105751 which bridges the splice site, and hybridizes to nucleotides555-574 of Genbank accession no. U72398, which encodes the unsplicedhuman bcl-x (bcl-x-beta). This target sequence corresponds to tennucleotides upstream of the 5′ splice site (positions 689-698 of Genbankaccession no. Z23115) and ten nucleotides of intron 1. TABLE 14Additional 2′-MOE oligonucleotides designed to alter splicing of humanbcl-xl Target Gen- bank SEQ Target Target acc. ID ISIS # Sequence Regionsite no NO: 106260 GTGGCCATCCAAGCTG coding/exon 630-649 Z23115 53 CGAT1L 106259 AAGTGGCCATCCAAGC coding/ 632-651 Z23115 54 TGCG exon 1L 106258GTAAGTGGCCATCCAA coding/ 634-653 Z23115 55 GCTG exon 1L 106257AGGTAAGTGGCCATCC coding/ 636-655 Z23115 56 AAGC exon 1L 106256TCAGGTAAGTGGCCAT coding/ 638-657 Z23115 57 CCAA exon 1L 106255ATTCAGGTAAGTGGCC coding/ 640-659 Z23115 58 ATCC exon 1L 106254TCATTCAGGTAAGTGG coding/ 642-661 Z23115 59 CCAT exon 1L 106253GGTCATTCAGGTAAGT coding/ 644-663 Z23115 60 GGCC exon 1L 106252GTGGTCATTCAGGTAA coding/ 646-665 Z23115 61 GTGG exon 1L 106251AGGTGGTCATTCAGGT coding/ 648-667 Z23115 62 AAGT exon 1L 106250CTAGGTGGTCATTCAG coding/ 650-669 Z23115 63 GTAA exon 1L 106249CTCTAGGTGGTCATTC coding/ 652-671 Z23115 64 AGGT exon 1L 26066ATCCAAGGCTCTAGGT coding/ 660-679 Z23115 65 GGTC exon 1L 22703CTGGATCCAAGGCTCT coding/ 664-683 Z23115 33 AGGT exon 1L 105751TGGTTCTTACCCAGCCexon 555-574 U72398 66 GCCG 1L/intron 1 689-698 Z2311526080 CTGGTTACACGACTCC 22783 mismatch 52 AGGT

[0265] These oligonucleotides were tested for their inhibitory effectson bcl-xl and bcl-xs mRNA levels in A549 cells, as detected by RPAanalysis. The results are shown in Table 15. Oligonucleotideconcentration was 200 nM. TABLE 15 Effect of antisense oligonucleotideson bcl-xs and bcl-xl Target site distance (from 5′ end of target site)upstream of SEQ ID % CONTROL bcl-xs/bcl-xl bcl-xl 5′ ISIS # NO bcl-xlFold difference splice site no oligo — 100 1 — 106260 53 66 12 68 10625954 96 15 66 106258 55 66 21 64 106257 56 78 13 62 106256 57 82 15 60106255 58 64 17 58 106254 59 80 13 56 106253 60 48 19 54 106252 61 72 2352 106251 62 57 25 50 106250 63 40 21 48 106249 64 47 23 46 26066 65 2437 38 22783 33 39 18 34 105751 66 34 45 Straddles splice site 26080 5277 2 Mismatch control

[0266] All of the oligonucleotides shown in the above table were able toincrease the bcl-xs/bcl-xl ratio by at least 12 fold and several(106249-106253, 26066, 22783 and 105751) increased the ratio over theprevious maximum of approximately 17-fold shown in Table 8. ISIS 105751and 26066 are highly preferred for increasing the bcl-xs/bcl-xl ratio to45-fold and 37-fold, respectively. This effect was dose-dependent, asmeasured for ISIS 26066 at doses from 50 to 400 nM. ISIS 26066 wastested and had no effect on bcl-x-beta (unspliced) levels.

[0267] Interestingly, all of the oligonucleotides that targeted a regionapproximately 15-54 nucleotides upstream of the 5′ splice site forbcl-xl are extremely potent redirectors of splicing, indicating thatthis region may be important for splice site selection. This issupported by the fact that mouse, rat and pig bcl-x sequences areidentical to the human sequence from nucleotide 654 (using the numberingof the human sequence in Genbank accession no. Z23115) through the 5′splice site at nucleotide 698. Accordingly, oligonucleotides targetingthe region extending approximately 60 nucleotides upstream (5′direction) from the splice site at nucleotide 698 are preferred.Oligonucleotides straddling the splice site are also preferred.

Example 44

[0268] Enhancement of Apoptosis in Androgen-Dependent Mouse ShionogiMammary Carcinoma Model

[0269] In the mouse Shionogi mammary carcinoma model, (Miyake et al.,1999), despite complete regression after castration, rapidly growingandrogen-dependent (AD) tumors recur after one month in a highlyreproducible manner, which provides a reliable time point to evaluatethe efficacy of agents that can delay time to androgen-independentprogression. The following study tested whether the adjuvant use ofantisense-bcl-xl antisense oligodeoxynucleotide (ODN) after castrationcould further delay progression to androgen independence.

[0270] Shionogi Tumor Growth

[0271] The Toronto subline of the transplantable SC-115 AD mouse mammarycarcinoma (Rennie et al., 1988) was used in all experiments. Shionogitumor cells were maintained in Dulbecco's modified Eagle medium (LifeTechnologies, Gaithersburg, Md.) supplemented with 5% fetal calf serum.For the in vivo study, approximately 5×10⁶ cells of the Shionogicarcinoma were injected subcutaneously into adult male DD/S strain mice.When tumors became 1 to 2 cm in diameter, usually 2 to 3 weeks afterinjection, castration was performed under methoxyflurane anesthesia.Details of the maintenance of mice, tumor stock and operative proceduresare described by Bruchovsky et al., 1978.

[0272] Antisense Oligonucleotides

[0273] ISIS 16009 was used as an antisense oligonucleotide to bcl-xl. Atwo-base bcl-xl mismatch ODN (5′-CTGG-ATCCAAGGATCGAGGT-3′, SEQ ID NO:67) was used as a control for in vitro studies. For in vivo studies, twocontrol oligonucleotides were used which do not have homology to anysequences through the basic local alignment search tool (BLAST) of theGenBank database (control oligonucleotide #1 —5′-TCTCCCGGCATGTGCCAT-3′;SEQ ID NO: 68) and control oligonucleotide #2—5′-TACCGTGTACGACCCTCT-3′;SEQ ID NO; 69).

[0274] Treatment of Cells with Antisense Oligonucleotides

[0275] Shionogi cells were treated with various concentrations ofantisense oligonucleotide after a preincubation for 20 min with 4 μg/mllipofection (Life Technologies) in OPTI-MEM medium (Life Technologies).Four hours after the start of incubation, the medium containingantisense oligonucleotide and lipofectin was replaced with standardculture medium described above.

[0276] Northern Blot Analysis

[0277] Total RNA was isolated from cultured Shionogi tumor cells andShionogi tumor tissues by the acid-guanidiniumthiocyanate-phenol-chloroform method. Poly A+ mRNA was purified fromtotal RNA using oligo dT-cellulose (Pharmacia Biotech, Upssala, Sweden).The electrophoresis, hybridization and washing conditions were aspreviously described (Miyake et al., supra.). Mouse bcl-xl and glycerol3-phosphate dehydrogenase (G3PDH) cDNA probes were generated by reversetranscription-polymerase chain reaction (RT-PCR) from total mouse brainRNA using the sense primer 5′-AG-TGCCATCAATGGCAACCCAT-3′ (SEQ ID NO: 70)and the antisense primer 5′-TCACTTCCGACTGAAGAGTGA-3′ (SEQ ID NO: 71) forbcl-xl, and the sense primer 5′-ATGGTGAAGGTCGGTGTGAACGGAT-3′ (SEQ ID NO:72) and antisense primer 5′-AAAGTTGTCATGGATGACCTT-3′ (SEQ ID NO: 73) forG3PDH. Density of bands for bcl-xl was normalized against that of G3PDHby densitometric analysis.

[0278] Western Blot Analysis

[0279] Samples containing equal amounts of protein (15 μg) from lysatesof the cultured Shionogi cells were electrophoresed on anSDS-polyacrylamide gel and transferred to a nitrocellulose filter. Thefilters were blocked in PBS containing 5% nonfat milk powder at 4° C.overnight and incubated for 1 hr with an anti-rat bcl-x mouse monoclonalantibody (Mab) (Transduction Laboratories, Mississauga, Canada),anti-rat β-tubulin mouse Mab (Chemicon International, Temecula, Calif.),anti-human caspase 1 rabbit polyclonal antibody (Upstate Biotechnology,Lake Placid, N.Y.), anti-human caspase 3 rabbit polyclonal antibody(Santa Cruz Biotechnology, Santa Cruz, Calif.) or anti-humanpoly(ADP-ribose) polymerase (PARP) mouse Mab (PharMingen, Missisauga,Canada) that reacts with the respective mouse target molecules. Thefilters were incubated for 30 min with horseradish peroxidase-conjugatedantimouse or rabbit IgG antibody (Amersham, Arlington Heights, Ill.),and specific proteins were detected using an enhganced chemiluminescenceWestern blotting analysis system (Amersham).

[0280] MTT Assay

[0281] The in vitro growth inhibitory effects of antisenseoligonucleotides and/or taxol on Shionogi tumor cells were asessed bythe MTT assay (Miyake et al., 1998). Briefly, 1×10⁴ cells were seeded ineach well of 96-well microtiter plates and allowed to attach overnight.Cells were then treated once daily with 500 nM oligonucleotide for 2days. Following oligonucleotide treatment, cells were treated withvarious concentrations of taxol. After a 48 hour incubation, 20 μl of 5mg/ml MTT (Sigma) in PBS was added to each well, followed by incubationfor 4 hr at 37° C. The formazan crystals were dissolved in DMSO. Theoptical density was determined with a microculture plate reader at 540nm. Absorbance values were normalized to the values obtained for thevehicle-treated cells to determine the percent of survival. Each assaywas performed in triplicate.

[0282] DNA Fragmentation Analysis

[0283] The nucleosomal DNA degradation was analyzed as describedpreviously with a minor modification (Miyake et al., 1998, supra.).Briefly, 1×10⁵ Shionogi tumor cells were seeded in 5-cm culture dishesand allowed to adhere overnight. After the treatment witholigonucleotide and/or taxol under the same schedule as described above,cells were harvested and lysed in a solution containing 10 mM Tris, pH7.4, 100 mM NaCl, 25 mM EDTA, 0.5% SDS. After centrifugation, thesupernatants were incubated with 300 μg/ml proteinase K for 5 hr at 65°C. and extracted with phenol-chloroform. The aqueous layer was treatedwith 0.1 vol of 3 M sodium acetate, and the DNA was precipitated with2.5 vol. Of 95% ethanol. Following treatment with 100 μg/ml RNase A for1 hr at 37° C., the sample was electrophoresed on a 2% agarose gel andstained with ethidium bromide.

[0284] Results

[0285] Northern blot analysis was used to characterize changes in bcl-xlmRNA expression in AD intact tumors before castration, regressing tumors4 and 7 days after castration, and AI recurrent tumors 28 days aftercastration. Bcl-xl mRNA expression is up-regulated 3-fold and 2-fold, 4and 7 days after castration, respectively, and remains 1.5-fold higherin AI tumors compared with AD intact tumors before castration (FIG. 1).The pattern of changes in bcl-xl expression in the Shiongi tumor modelduring AI progression is similar to that in the clinical disease(Krajewski et al., 1996) and therefore supports the use of this model toevaluate the effect of adjuvant antisense bcl-xl therapy on progressionto androgen independence.

[0286] Northern blot analysis was also used to evaluate the effect oftreatment with antisense bcl-xl oligonucleotide on bcl-xl mRNAexpression. Daily treatment of Shionogi cells with antisense bcl-xloligonucleotide (50, 100, 500 or 1,000 nM) for 2 days decreased bcl-xlmRNA levels by 0%, 7%, 61% or 89%, respectively, whereas bcl-xl mRNAexpression was not affected by the 2-base mismatch controloligonucleotide at any of the employed concentrations (FIG. 2). Toexaminer whether reduced bcl-xl mRNA levels induced by antisenseoligonucleotide is accompanied by a corresponding decrease in proteinlevels, Western blot analysis was used to analyze changes in bcl-xlprotein levels in Shionogi cells following daily treatment withantisense bcl-xl oligonucleotide for 5 consecutive days. Dose-dependentinhibition of bcl-xl protein levels was observed with antisense bcl-xloligonucleotide but not with mismatch control oligonucleotide treatment.

[0287] Male mice bearing Shionogi tumors 1 to 2 cm in diameter werecastrated and randomly selected for treatment with ISIS 16009 vs.control oligonucleotide #1. Mean tumor volume was similar in both groupsat the beginning of treatment. Beginning 1 day post castration, 12.5mg/kg ISIS 16009 was administered once daily by i.p. injection for 40days. ISIS 16009 treatment significantly delayed recurrence of AI tumorscompared with control oligonucleotide #1 treatment (FIG. 3). During anobservation period of 7 weeks postcastration, AI tumors recurred in allmice after a median of 38 or 31 days postcastration in ISIS 16009 orcontrol ODN#1 treatment group, respectively. Moreover, by 7 weekspostcastration, mean tumor volume in mice treated with ISIS 16009 was30% smaller than in mice treated with control oligonucleotide #1.

[0288] The effects of in vivo antisense oligonucleotide treatment ofbcl-xl mRNA expression in Shionogi tumors was then examined by Northernblotting. Beginning 1 day postcastration, each of 3 tumor-bearing micewas administered 12.5 mg/kg ISIS 16009 or control oligonucleotide #1.I.p. once daily, and tumor tissues were harvested for mRNA extraction 4days postcastration. ISIS 16009 treatment resulted in 58% reduction inbcl-xl mRNA levels in Shionogi tumors compared with mismatch controlODN#1-treated tumors (FIG. 4).

[0289] To determine whether antisense bcl-xl oligonucleotide alsoenhances the cytotoxic effect of taxol, Shionogi cells were treated with500 nM ISIS for 2 days and then incubated with various concentrations oftaxol for 2 days. ISIS 16009, significantly enhanced taxolchemosensitivity, reducing the IC₅₀ of taxol from 100 nM to 50 nM.

[0290] A DNA fragmentation assay was performed to compare the effects of500 nM ISIS 16009 plus 10 nM taxol treatment on induction of apoptosisafter the same treatment schedule as described above. The characteristicapoptotic DNA ladder was observed after taxol treatment combined withISIS 16009.

[0291] ISIS 16009 inhibited expression of bcl-xl mRNA and protein in adose-dependent and sequence-specific manner. In in vivo experiments,administration of ISIS 16009 reduced bcl-xl mRNA levels in Shionogitumors and delayed time to AI progression compared with that of controloligonucleotide. These findings indicate that bcl-xl is a suitablemolecular target for antisense oligonucleotide therapy.

Example 45

[0292] Promotion of Apoptosis in Glioblastoma Cells by ISIS 16009

[0293] Bcl-xl expression in glioblastomas has been linked to diseaseprogression and treatment resistance (Bruggers et al., J. Pediatr.Hematol. Oncol. 21:19-25, 1999; Krajewski et al., Am. J. Pathol.150:805-814, 1997). The following studies were performed to determinewhether antisense-mediated reduction of bcl-xl could facilitateapoptosis and reduce the chemoresistance of human glioma cells.

[0294] Cell Culture

[0295] The human glioblastoma cell line MO59K (American type CultureCollection, Manassas, Va.) was cultured in DMEM/F12 medium (Gibco BRL,Paisley, UK) supplemented with 8% fetal calf serum and 100 units/mlpenicillin, 100 μg/ml streptomycin and 0.25 μg/ml Amphotericin B (GibcoBRL) in a fully humidified 5% CO₂/95% ambient air atmosphere at 37° C.

[0296] Taxol

[0297] A 1 mg/ml stock solution of taxol in saline (Bristol-MyersSquibb, Princeton, N.J., a microtubule stabilizing agent frequently usedin treatment regimens for glioblastomas, was diluted to the requiredconcentration with 0.9% saline immediately before use.

[0298] Delivery of Oligonucleotides

[0299] Cells were seeded at a density of 0.25×10⁶ in 6-well plates 24hours before oligonucleotide treatment. Cultures were then incubated for4 hours at 37° C. with 200 nM oligonucleotide in the presence of 10 μlLipofectin as an uptake enhancer, according to the manufacturer'sprotocol. After incubation, the oligonucleotide-lipofectin mixture wasreplaced with complete medium and cells were cultured as describedabove.

[0300] Western Blot Analysis

[0301] Proteins were extracted in lysis buffer (25 mM Tris HCl, pH 7.4,150 mm NaCl, 1% Triton X-100, 5 μg/ml leupeptin, aprotinin andpepstatin, 1 μg/ml benzamidine HCl, 1 mM sodium orthovanadate and 1 mMPMSF (all from Sigma), and protein concentrations were determined. Equalamounts of proteins (10 μg) were subjected to electrophoresis on 12% SDSpolyacrylamide gels and transferred to PVDF membranes (Millipore,Bedford, Mass.) in PBS, then incubated with rabbit polyclonal antibodiesagains bcl-x (Transduction Laboratories) or actin (Sigma) for 1 hour in0.2% I-block. Membranes were washed, then incubated with optimallydiluted alkaline phosphatase-conjugated goat anti-rabit IgG (Tropix) in0.2% I-block followed by detection of reactive bands bychemoluminescence (CSPD substrate, Tropix).

[0302] Viability Assays

[0303] Viability assays were performed in 96-well plates (Costar) oncells cultured as described above. Cells were incubated with taxol in 10μl medium to a final concentration of 2.5 nM. Controls contained 10 μlmedium with the corresponding amount of saline. Viability was determinedwith the calorimetric WST-1 assay (Boehringer mannheim, Indianapolis,Ind.). and measured using a Dynatech MR-7000 ELISA reader acording tothe manufacturer's instructions.

[0304] Flow Cytometry

[0305] Cells were harvested 48 h after addition of antisenseoligonucleotides, corresponding to 24 h after initiation of taxoltreatment, and processed for cell cycle analysis with the cycleTESTpluskit (Becton Dickinson, Franklin Lakes, N.J.) according to themanufacturer's recommendations. Gates were set to exclude sub-cellularparticles and doublets and at least 1.5×10⁴ events analyzed on aFACScalibur (Becton Dickinson) with an argon laser tuned at 488 nm.

[0306] Statistical Analysis

[0307] The statistical significance of differences in viability wasdetermined using the Mann-whitney U test. P values less than 0.05 wereconsidered to be of statistical significance.

[0308] Results

[0309] Treatment of MO59K cells with 200 nM ISIS 16009 for 4 hoursreduced bcl-xl expression after 24 hours. Bcl-xl expression wasundetectable after 48 hours as determined by Western blotting. Mismatchcontrol oligonucleotide (ISIS 16967) had a much less pronounced effecton bcl-xl levels (Max. increase of 12.5% ±SD) compared to saline treatedcontrols. Western blot analysis demonstrated no changes in bcl-xs levelsor expression of bcl-2, bax, bad or bak proteins after ISIS 16009treatment.

[0310] To determine whether lower bcl-xl protein levels sensitized MO59Kcells to apoptosis-inducing agents, cells were treated with taxol at aconcentration of 2.5 nM 24 hours after oligonucleotide treatment. Taxoltreatment alone reduced viability in saline and mismatched controlgroups by 25%±SD and 28%±SD after 24 hours, and 37%±SD and 44.5%±SDafter 48 hours, respectively (FIG. 5). In bcl-xl antisense treatedgroups, increased cytotoxicity was observed 24 hours after addition oftaxol compared to saline (25%±SD) and mismatch (28%±SD) groups,increasing to 66.5%±SD and 83%±SD after 48 hours, respectively. After 4days, cell viability in taxol and antisense bcl-xl treated cultures wasreduced to 17%±SD compared to 63%±SD and 55.5%±SD in the saline andmismatch groups treated with taxol, respectively (FIG. 5). Thedifference between antisense bcl-xl treated cultures and mismatch groupswas statistically significant (p<0.005).

[0311] Treatment of MO59K cells with 2.5 nM taxol induced apoptosisafter 24 hours as as assessed by cells possessing sub-G0/G1 DNA contentin FACS cell cycle analysis. To examine the mechanism of enhancedcytotoxicity induced by bcl-xl antisense treatemnt in taxol stimulatedcultures, flow cytometric analyses were performed 24 hours after taxoladministration. Compared to the mismatch control oligonucleotide, ISIS16009 treatment increased the number of cells with sub-G0/G1 content. Aconcomitant reduction in the number of cells in the G0/G1 phase wasseen, suggesting that the increased apoptosis induced by down-regulationof bcl-xl is cell cycle specific. Thus, bcl-xl antisenseoligonucleotides improve the chemosensitivity of gliomas. The use ofantisense olgonucleotides which target bcl-xl in enhancing thechemosensitivity of any cancer cell or tumor type is within the scope ofthe present invention.

Example 46

[0312] Sensitization of Leukemia Cells to Chemotherapeutic Drugs bybcl-xl Antisense Oligonucleotide

[0313] Bcl-xl levels are particularly important for the regulation ofimmature T lymphocyte (thymocyte) apoptosis. Acute lymphocytic leukemia(ALL) is the most common malignancy of childhood. T-ALL are malignancieswith cells arrested in various stages of thymocyte differentiation. BothT-ALL and normal thymocytes are rapidly proliferating cells. Theaccumulation of cells that is characteristic of leukemia reflects thiscell growth and a lack of apoptosis. Most normal thymocytes undergoapoptosis, never maturing to peripheral T cells. Physiologic thymocyteapoptosis depends upon an orchestrated modulation of bcl-2 and relatedproteins, including bcl-xl and bax (Ma et al., Proc. Natl. Acad. Sci.U.S.A., 1997, 92, 4763-4767; Nakayama et al., Science, 1993, 261,1584-1588).

[0314] Since T-cell acute lymphocytic leukemia (T-ALL) cells aremalignant versions of immature T lymphocytes, the effects of antisenseoligonucleotides to bcl-x on the survival and chemosensitivity of CEMcells, a T-ALL cell line, were examined.

[0315] Patient Materials

[0316] All 69 T-ALL specimens originated from previously untreatedpatients enrolled in the Pediatric Oncology Group (POG) trials.

[0317] Cell Lines and Culture

[0318] CEM-C7 cells (Norman et al., 1978), a glucocortocoid sensitiveclone of the human T-lymphoblastic cell line CEM, were cultured between5×10⁴ to 10⁶ cells/ml in RPMI 1640 medium containing 10%heat-inactivated fetal bovine serum (FBS), streptomycin, penicillin G,15 mM HEPES, 2 mM sodium pyruvate, at 37° C. in a humid, 5% CO₂incubator. Viable cell counts were performed by propidium iodidestaining and flow cytometry.

[0319] Drugs

[0320] Doxorubicin and dexamethasone (Calbiochem, San Diego, Calif.)were dissolved in water and further diluted in cell culture media toobtain the desired final concentrations.

[0321] Oligonucleotide Treatment of Cells

[0322] CEM-C7 cells were electroporated with the indicatedconcentrations of oligonucleotides at 150 volts, 1,700 microFaraday(μF), and a resistance setting of 5 with a BTX electroporation apparatus(Genetronics, San Diego, Calif.) in 2-mm gap cuvettes containing 5×10⁶cells in 200 μl RPMI plus 20% FBS on ice. Cells were left on ice for 10minutes, then plated in 5 ml complete media.

[0323] Immunoblot Assays

[0324] Postnuclear detergent lysates were prepared as described by Reedet al. (Cancer Res., 1991, 51, 6529-6538), and the protein concentrationwas determined using a Bradford method protein assay kit (Bio-Rad,Hercules, Calif.). Protein (20 μg) was loaded in each lane, followed byelectrophoresis on a 12% sodium dodecyl sulfate-polyacrylamide gel,followed by immunoblotting (Winston et al., 1993). The primary antibodywas a 0.4 μg/ml dilution of the mouse monoclonal anti-human bcl-2 (clone124, Dako, Carpinteria, Calif.), a 4.0 μg/ml dilution of the rabbitpolyclonal anti-human bcl-xs/l (Dako), or a 0.3 μg/ml dilution of rabbitpolyclonal anti-human Bax (N-20, Santa Cruz Biotechnology, Santa Cruz,Calif.). For development and STORM fluorescent imaging, the secondaryantibody was peroxidase-conjugated sheep anti-mouse or donkeyanti-rabbit (Amersham, Little Chalfort, UK). Chemiluminescence imagingand quantification were preformed using the STORM instrument andImageQuant software (Molecular Dynamics, Sunnyvale, Calif.).

[0325] Statistical Methods

[0326] To determine if synergy existed between the bcl-x and thechemotherapeutic drugs dexamethasone or doxorubicin, the median-effectprinciple of Chou (“The median-effect principle and the combinationindex for quantification of synergism and antagonism. In Synergism andAntagonism in Chemotherapy, T.-C. Chou and D. Rideout, eds., AcademicPress, New York, pp. 61-102) was used to determine dose-effectparameters for the drugs individually and for the combinations of bcl-xantisense plus dexamethasone or bcl-x antisense plus doxorubicin. Thismethod analyzes the shape of the drug dose-response curves for eachdrug, combination of drugs and quantitates synergism/antagonism atdifferent concentrations. Median effect computer software (CalcuSyn forWindows, Biosoft, Ferguson, Mo.) was used to generate the combinationindex (CI), where a CI value of <1, =1, >1 indicates synergism (i.e.,the effect of drug combination is greater than anticipated from theadditive effect of the individual agents), additive effect, andantagonism, respectively.

[0327] Results

[0328] The levels of bcl-2, bcl-x and bax were determined in extractsfrom 69 cases of T-ALL by immunoblot assay. Most cases expressed allthree proteins at detectable levels. Normal thymus was included in allblots as an internal control, and, after normalization to thymus, themean+/−one standard deviation was 3.1±6.7 for bcl-2, 0.3±0.3 for bcl-xland 1.3±1.1 for bax. The ranges for bcl-2, bcl-xl and bax were 0.1 to54, <0.1 to 1.5, and 0.2 to 6.0 times the levels in thymus,respectively. Table 16 shows this data in category form. Relative tothymus, the majority of T-ALL specimens have higher bcl-2, lower bcl-xand similar levels of bax. Also, the levels of bcl-2 and bcl-x variedmore than the level of bax. No bcl-xs was detected in extracts from theT-ALL cells or thymus. TABLE 16 Bcl-2 related proteins in primary T-ALLFold over thymus Bcl-2 Bcl-x Bax ≧3.1  20 (29%) 0 5 (7%)      1.1-3.0 22(32%) 2 (3%) 23 (33%)    0.26-1.0  20 (29%) 29 (40%) 39 (56%)     0.11-0.25 4 (6%) 23 (33%) 2 (3%) ≦0.10 3 (4%) 15 (22%) 0

[0329] The effects of bcl-x antisense oligonucleotides on CEM-C7 cells,a glucocorticoid-sensitive clone of CEM cells, was examined. This cellline expresses levels of bcl-2, bcl-x and bax that are 0.25×, 1.1×, and0.5× the level of normal thymus, respectively. These levels of bcl-2,bcl-x and bax put this cell line in about the 15^(th), 95^(th), and50^(th) percentile rank, respectively, of all T-ALL cases (Table 16).

[0330] The bcl-x antisense oligonucleotide, ISIS 15999 (SEQ ID NO: 21),targets the bcl-x mRNA at 1 to 20 base pairs downstream of thetranslation start site. Twenty hours after electroporation with theindicated concentration of antisense oligonucleotide to bcl-x, the CEMcells showed a dose dependent decrease in viability (FIG. 7A). At thehighest concentration, the treated cells had a cell count that was 40%less than the control antisense (ISIS 16971; 5′-TCACATTGGCGCTTAGCCGT-3′,SEQ ID NO: 74) treated cells. At this concentration, the viable cellcount for the control antisense treated cells started to decrease,indicating some non-specific toxicity. At the lower concentrations, thecontrol-treated cells had the same viable cell counts as the mockelectroporated cells.

[0331] The level of bcl-xl protein in the CEM cells also showed a dosedependent decrease after treatment with antisense oligonucleotide tobcl-x (FIG. 7B). The effect is specific, since treatment with thecontrol oligonucleotide had no effect on the bcl-xl levels, and sincebcl-2 protein levels did not decrease with bcl-x antisense treatment,even at the highest concentration.

[0332] Bcl-x antisense treatment not only decreased the viable cellcounts at 24 hours, it also caused the CEM cells to grow less well forup to 72 hours (FIG. 8). This reduced growth reflected continuedincreased cell death by the bcl-x antisense-treated cells. By propidiumiodide exclusion testing, the bcl-x antisense treated cells showed only33% and 46% viability at 48 and 72 hours, respectively. The controltreated cells showed 70% and 82% viability at 48 and 72 hours,respectively.

[0333] Treatment of CEM cells with bcl-x antisense oligonucleotide makesthe cells more sensitive to killing by doxorubicin or dexamethasone(FIGS. 9A-B). These, or similar drugs, are used frequently for acutelymphocytic leukemia treatment. The use of any of these therapeuticagents in combination with one or more antisense oligonucleotides tobcl-x is within the scope of the present invention. For theseexperiments, the CEM cells were electroporated with either the bcl-xantisense or control oligonucleotides, cultured for one day, thentreated with the indicated concentrations of drug. Analysis by themedian-effect principle revealed a combination index (CI) of <0.3 forthe three highest doses of dexamethasone and 0.3 to 0.4 for the twolower doses of dexamethasone (FIG. 10). A CI of <0.3 is classified as astrong degree of synergism, and 0.3 to 0.7 is classified as synergism.In contrast, the combination of bcl-x antisense and doxorubicin did notreveal synergy. For the doses tested of doxorubicin, the CI ranged from0.7 to 1.3.

[0334] It is believed that these results are the first demonstrationthat bcl-x antisense oligonucleotides can decrease the survival ofleukemia cells and increase their sensitivity to chemotherapeutic drugs.The antisense treatment is specific, since it shows dose dependence,does not affect bcl-2 levels, and a scrambled version does not affectbcl-x levels or apoptosis. The increased sensitivity to chemotherapeuticdrugs was additive for doxorubicin and synergistic for dexamethasone.

[0335] The examples provided above relating to the chemosensitization ofglioblastoma cells and leukemia cells by to an apoptotic stimulus byantisense oligonucleotides to bcl-xl are intended to be illustrativerather than limiting. These antisense oligonucleotides can also be usedto chemosensitize other types of cancer cells, including, but notlimited to, lung carcinoma, melanoma and neuroblastoma.

Example 47

[0336] Antisense Inhibition of bcl-xl in the Liver

[0337] A single injection of mouse Fas-specific monoclonal antibody Jo-2(Pharmingen) into mice will induce extensive hepatocyte apoptosis,hemorrhage in the liver, increases in serum aminotransferase levels andanimal death at high antibody doses (Ogasawara et al., Nature364:806-809, 1993). To demonstrate antisense inhibition of bcl-xl mRNAand protein expression in the liver, mice were pre-dosed with 50 mg/kgbcl-xl antisense oligonucleotide ISIS 16009, 50 mg/kg control antisenseoligonucleotide ISIS 20292 or saline injected intraperitoneally everyother day for 8 days (4 injections total). One day later, 3 μg Jo-2antibody was injected intraperitoneally to induce fas activation. Twelvehours later, animals were sacrificed and total RNA extracted from liverusing an RNeasy kit (Qiagen, Valencia, Calif.). RNase protection assay(RPA) was performed according to the manufacturer's instructions(Pharmingen, San Diego, Calif.). RPA template mApo-3 and a customtemplate (Pharmingen) were used as probes. Twenty μg total RNA wasanalyzed on 6% polyacrylamide gels. The bcl-xl antisense oligonucleotideinhibited bcl-xl mRNA expression in mouse liver greater than 80%, whilethe expression of other mRNA species (bak, bax, bad, L32) were notaffected. Bcl-xl protein expression was also inhibited by greater than80% as determined by SDS-polyacrylamide gel electrophoresis and Westernblotting of liver extracts.

Example 48

[0338] Effect of Reduction of bcl-xl Expression in Vivo on Survival

[0339] To determine whether reduction of bcl-xl expression sensitizesmice to fas antibody-induced death, 30 mice were pre-dosedintraperitoneally every other day for 8 days as follows: 50 mg/kganti-bcl-xl antisense oligonucleotide ISIS 16009 (10 mice), controloligonucleotide ISIS 20292 (10 mice) or saline (10 mice). One day later,3 μg fas antibody was injected intraperitoneally to induce fasactivation. Survival was then monitored. None of the mice treated withbcl-xl antisense oligonucleotide survived beyond 13 hours after fasantibody administration (FIG. 11). In contrast, 8 of the animalsadministered the control oligonucleotide were still alive after 13hours.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 74 <210> SEQ ID NO 1<211> LENGTH: 926 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (135)..(836) <308> DATABASEACCESSION NUMBER: L20121 Genbank <309> DATABASE ENTRY DATE: 1994-07-26<400> SEQUENCE: 1 gaatctcttt ctctcccttc agaatcttat cttggctttg gatcttagaagagaatcact 60 aaccagagac gagactcagt gagtgagcag gtgttttgga caatggactggttgagccca 120 tccctattat aaaa atg tct cag agc aac cgg gag ctg gtg gttgac ttt 170 Met Ser Gln Ser Asn Arg Glu Leu Val Val Asp Phe 1 5 10 ctctcc tac aag ctt tcc cag aaa gga tac agc tgg agt cag ttt agt 218 Leu SerTyr Lys Leu Ser Gln Lys Gly Tyr Ser Trp Ser Gln Phe Ser 15 20 25 gat gtggaa gag aac agg act gag gcc cca gaa ggg act gaa tcg gag 266 Asp Val GluGlu Asn Arg Thr Glu Ala Pro Glu Gly Thr Glu Ser Glu 30 35 40 atg gag accccc agt gcc atc aat ggc aac cca tcc tgg cac ctg gca 314 Met Glu Thr ProSer Ala Ile Asn Gly Asn Pro Ser Trp His Leu Ala 45 50 55 60 gac agc cccgcg gtg aat gga gcc act gcg cac agc agc agt ttg gat 362 Asp Ser Pro AlaVal Asn Gly Ala Thr Ala His Ser Ser Ser Leu Asp 65 70 75 gcc cgg gag gtgatc ccc atg gca gca gta aag caa gcg ctg agg gag 410 Ala Arg Glu Val IlePro Met Ala Ala Val Lys Gln Ala Leu Arg Glu 80 85 90 gca ggc gac gag tttgaa ctg cgg tac cgg cgg gca ttc agt gac ctg 458 Ala Gly Asp Glu Phe GluLeu Arg Tyr Arg Arg Ala Phe Ser Asp Leu 95 100 105 aca tcc cag ctc cacatc acc cca ggg aca gca tat cag agc ttt gaa 506 Thr Ser Gln Leu His IleThr Pro Gly Thr Ala Tyr Gln Ser Phe Glu 110 115 120 cag gta gtg aat gaactc ttc cgg gat ggg gta aac tgg ggt cgc att 554 Gln Val Val Asn Glu LeuPhe Arg Asp Gly Val Asn Trp Gly Arg Ile 125 130 135 140 gtg gcc ttt ttctcc ttc ggc ggg gca ctg tgc gtg gaa agc gta gac 602 Val Ala Phe Phe SerPhe Gly Gly Ala Leu Cys Val Glu Ser Val Asp 145 150 155 aag gag atg caggta ttg gtg agt cgg atc gca gct tgg atg gcc act 650 Lys Glu Met Gln ValLeu Val Ser Arg Ile Ala Ala Trp Met Ala Thr 160 165 170 tac ctg aat gaccac cta gag cct tgg atc cag gag aac ggc ggc tgg 698 Tyr Leu Asn Asp HisLeu Glu Pro Trp Ile Gln Glu Asn Gly Gly Trp 175 180 185 gat act ttt gtggaa ctc tat ggg aac aat gca gca gcc gag agc cga 746 Asp Thr Phe Val GluLeu Tyr Gly Asn Asn Ala Ala Ala Glu Ser Arg 190 195 200 aag ggc cag gaacgc ttc aac cgc tgg ttc ctg acg ggc atg act gtg 794 Lys Gly Gln Glu ArgPhe Asn Arg Trp Phe Leu Thr Gly Met Thr Val 205 210 215 220 gcc ggc gtggtt ctg ctg ggc tca ctc ttc agt cgg aaa tga 836 Ala Gly Val Val Leu LeuGly Ser Leu Phe Ser Arg Lys 225 230 ccagacactg accatccact ctaccctcccacccccttct ctgctccacc acatcctccg 896 tccagccgcc attgccacca ggagaacccg926 <210> SEQ ID NO 2 <211> LENGTH: 233 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <400> SEQUENCE: 2 Met Ser Gln Ser Asn Arg Glu Leu Val ValAsp Phe Leu Ser Tyr Lys 1 5 10 15 Leu Ser Gln Lys Gly Tyr Ser Trp SerGln Phe Ser Asp Val Glu Glu 20 25 30 Asn Arg Thr Glu Ala Pro Glu Gly ThrGlu Ser Glu Met Glu Thr Pro 35 40 45 Ser Ala Ile Asn Gly Asn Pro Ser TrpHis Leu Ala Asp Ser Pro Ala 50 55 60 Val Asn Gly Ala Thr Ala His Ser SerSer Leu Asp Ala Arg Glu Val 65 70 75 80 Ile Pro Met Ala Ala Val Lys GlnAla Leu Arg Glu Ala Gly Asp Glu 85 90 95 Phe Glu Leu Arg Tyr Arg Arg AlaPhe Ser Asp Leu Thr Ser Gln Leu 100 105 110 His Ile Thr Pro Gly Thr AlaTyr Gln Ser Phe Glu Gln Val Val Asn 115 120 125 Glu Leu Phe Arg Asp GlyVal Asn Trp Gly Arg Ile Val Ala Phe Phe 130 135 140 Ser Phe Gly Gly AlaLeu Cys Val Glu Ser Val Asp Lys Glu Met Gln 145 150 155 160 Val Leu ValSer Arg Ile Ala Ala Trp Met Ala Thr Tyr Leu Asn Asp 165 170 175 His LeuGlu Pro Trp Ile Gln Glu Asn Gly Gly Trp Asp Thr Phe Val 180 185 190 GluLeu Tyr Gly Asn Asn Ala Ala Ala Glu Ser Arg Lys Gly Gln Glu 195 200 205Arg Phe Asn Arg Trp Phe Leu Thr Gly Met Thr Val Ala Gly Val Val 210 215220 Leu Leu Gly Ser Leu Phe Ser Arg Lys 225 230 <210> SEQ ID NO 3 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400>SEQUENCE: 3 cgggttctcc tggtggcaat 20 <210> SEQ ID NO 4 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 4cagtgtctgg tcatttccga 20 <210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 5 agcccagcagaaccacgccg 20 <210> SEQ ID NO 6 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Antisense Oligonucleotide <400> SEQUENCE: 6 gttgaagcgt tcctggccct 20<210> SEQ ID NO 7 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 7 cagtgccccg ccgaaggaga 20 <210> SEQ IDNO 8 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 8 tcgcctgcct ccctcagcgc 20 <210> SEQ IDNO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 9 cagtggctcc attcaccgcg 20 <210> SEQ IDNO 10 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 10 attcagtccc ttctggggcc 20 <210> SEQ IDNO 11 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 11 aaagtcaacc accagctccc 20 <210> SEQ IDNO 12 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 12 ccggttgctc tgagacattt 20 <210> SEQ IDNO 13 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 13 accagtccat tgtccaaaac 20 <210> SEQ IDNO 14 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 14 gaagggagag aaagagattc 20 <210> SEQ IDNO 15 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 15 tcattcacta cctgttcaaa 20 <210> SEQ IDNO 16 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 16 agcccaccag aaggaccccg 20 <210> SEQ IDNO 17 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 17 cagtggctct caccgcatcg 20 <210> SEQ IDNO 18 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 18 cagcccgcct gcgaaggaga 20 <210> SEQ IDNO 19 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 19 agcgcagaac caccacgccg 20 <210> SEQ IDNO 20 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 20 taatagggat gggctcaacc 20 <210> SEQ IDNO 21 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 21 tcccggttgc tctgagacat 20 <210> SEQ IDNO 22 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 22 gggcctcagt cctgttctct 20 <210> SEQ IDNO 23 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 23 tccatctccg attcagtccc 20 <210> SEQ IDNO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 24 aggtgccagg atgggttgcc 20 <210> SEQ IDNO 25 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 25 agtggctcca ttcaccgcgg 20 <210> SEQ IDNO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 26 cttgctttac tgctgccatg 20 <210> SEQ IDNO 27 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 27 gccggtaccg cagttcaaac 20 <210> SEQ IDNO 28 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 28 ctgttcaaag ctctgatatg 20 <210> SEQ IDNO 29 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 29 taccccatcc cggaagagtt 20 <210> SEQ IDNO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 30 aaaggccaca atgcgacccc 20 <210> SEQ IDNO 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 31 ctacgctttc cacgcacagt 20 <210> SEQ IDNO 32 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 32 tccaagctgc gatccgactc 20 <210> SEQ IDNO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 33 ctggatccaa ggctctaggt 20 <210> SEQ IDNO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 34 ccagccgccg ttctcctgga 20 <210> SEQ IDNO 35 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 35 tagagttcca caaaagtatc 20 <210> SEQ IDNO 36 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 36 agcgttcctg gccctttcgg 20 <210> SEQ IDNO 37 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 37 gtcatgcccg tcaggaacca 20 <210> SEQ IDNO 38 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 38 tgagcccagc agaccacgc 19 <210> SEQ IDNO 39 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 39 gagggtagag tggatggtca 20 <210> SEQ IDNO 40 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 40 ggaggatgtg gtggagcaga 20 <210> SEQ IDNO 41 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 41 gacatccctt tccccctcgg 20 <210> SEQ IDNO 42 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 42 Asp Glu Val Asp 1 <210> SEQ ID NO 43<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400>SEQUENCE: 43 ctccgatgtc ccctcaaagt 20 <210> SEQ ID NO 44 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 44tcacgttggc gcttagccat 20 <210> SEQ ID NO 45 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 45 caaaagtatcccagccgccg 20 <210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Antisense Oligonucleotide <400> SEQUENCE: 46 gccgccgttc tcctggatcc 20<210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 47 gttcctggcc ctttcggctc 20 <210> SEQ IDNO 48 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 48 caggaaccag cggttgaagc 20 <210> SEQ IDNO 49 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 49 ccggccacag tcatgcccgt 20 <210> SEQ IDNO 50 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 50 tgtagcccag cagaaccacg 20 <210> SEQ IDNO 51 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 51 cgacacgtac ctctcgcatt 20 <210> SEQ IDNO 52 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 52 ctggttacac gactccaggt 20 <210> SEQ IDNO 53 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 53 gtggccatcc aagctgcgat 20 <210> SEQ IDNO 54 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 54 aagtggccat ccaagctgcg 20 <210> SEQ IDNO 55 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 55 gtaagtggcc atccaagctg 20 <210> SEQ IDNO 56 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 56 aggtaagtgg ccatccaagc 20 <210> SEQ IDNO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 57 tcaggtaagt ggccatccaa 20 <210> SEQ IDNO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 58 attcaggtaa gtggccatcc 20 <210> SEQ IDNO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 59 tcattcaggt aagtggccat 20 <210> SEQ IDNO 60 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 60 ggtcattcag gtaagtggcc 20 <210> SEQ IDNO 61 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 61 gtggtcattc aggtaagtgg 20 <210> SEQ IDNO 62 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 62 aggtggtcat tcaggtaagt 20 <210> SEQ IDNO 63 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 63 ctaggtggtc attcaggtaa 20 <210> SEQ IDNO 64 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 64 ctctaggtgg tcattcaggt 20 <210> SEQ IDNO 65 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 65 atccaaggct ctaggtggtc 20 <210> SEQ IDNO 66 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 66 tggttcttac ccagccgccg 20 <210> SEQ IDNO 67 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 67 ctggatccaaggatcgaggt 20 <210> SEQ IDNO 68 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 68 tctcccggcatgtgccat 18 <210> SEQ ID NO69 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400>SEQUENCE: 69 taccgtgtacgaccctct 18 <210> SEQ ID NO 70 <211> LENGTH: 22<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: PCR Primer <400> SEQUENCE: 70 agtgccatcaatggcaacccat22 <210> SEQ ID NO 71 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer<400> SEQUENCE: 71 tcacttccgactgaagagtga 21 <210> SEQ ID NO 72 <211>LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 72atggtgaaggtcggtgtgaacggat 25 <210> SEQ ID NO 73 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR Primer <400> SEQUENCE: 73 aaagttgtcatggatgacctt 21<210> SEQ ID NO 74 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 74 tcacattggcgcttagccgt 20

What is claimed is:
 1. An antisense compound 8 to 30 nucleobases inlength targeted to a 3′-untranslated region, a stop codon region, acoding region, or a 5′-untranslated region of a nucleic acid moleculeencoding a human bcl-x of SEQ ID NO:1, wherein said antisense compoundmodulates the expression of human bcl-x.
 2. The antisense compound ofclaim 1 which is an antisense oligonucleotide.
 3. The antisense compoundof claim 2 wherein the antisense oligonucleotide comprises at least onemodified internucleoside linkage.
 4. The antisense compound of claim 3wherein the modified internucleoside linkage of the antisenseoligonucleotide is a phosphorothioate linkage, a morpholino linkage or apeptide-nucleic acid linkage.
 5. The antisense compound of claim 2wherein the antisense oligonucleotide comprises at least one modifiedsugar moiety.
 6. The antisense compound of claim 5 wherein the modifiedsugar moiety of the antisense oligonucleotide is a 2′-O-methoxyethylsugar moiety or a 2′-dimethylaminooxyethoxy sugar moiety.
 7. Theantisense compound of claim 5 wherein substantially all sugar moietiesof the antisense oligonucleotide are modified sugar moieties.
 8. Theantisense compound of claim 3 wherein the antisense oligonucleotidecomprises at least one modified nucleobase.
 9. The antisense compound ofclaim 8 wherein the modified nucleobase of the antisense oligonucleotideis a 5-methylcytosine.
 10. The antisense compound of claim 8 whereineach modified nucleobase of the antisense oligonucleotide is a5-methylcytosine.
 11. The antisense compound of claim 1 which is achimeric oligonucleotide.
 12. A pharmaceutical composition comprisingthe antisense compound of claim 1 and a pharmaceutically acceptablecarrier or diluent.
 13. The pharmaceutical composition of claim 12further comprising a colloidal dispersion system.
 14. The pharmaceuticalcomposition of claim 12 wherein the antisense compound is an antisenseoligonucleotide.
 15. The antisense compound of claim 1 which is targetedto bcl-xl.
 16. The antisense compound of claim 1 which alters the ratioof bcl-x isoforms expressed by a cell or tissue.
 17. The antisensecompound of claim 16 which increases the ratio of bcl-xl to bcl-xsexpressed.
 18. The antisense compound of claim 16 which decreases theratio of bcl-xl to bcl-xs expressed.
 19. A method of inhibiting theexpression of bcl-x in human cells or tissues comprising contacting saidcells or tissues with the antisense compound of claim 1 so thatexpression of bcl-x is inhibited.
 20. A method of treating an animalhaving a disease or condition characterized by a reduction in apoptosiscomprising administering to said animal a prophylactically ortherapeutically effective amount of the antisense compound of claim 1 sothat apoptosis is increased.
 21. The method of claim 20 wherein theantisense compound is targeted to a nucleic acid molecule encodingbcl-xl and which preferentially inhibits the expression of bcl-xl. 22.The pharmaceutical composition of claim 12 further comprising achemotherapeutic agent for the treatment of cancer.
 23. A method ofsensitizing a cell to an apoptotic stimulus comprising treating the cellwith the antisense compound of claim
 1. 24. The method of claim 23wherein the apoptotic stimulus is radiation.
 25. The method of claim 24wherein the radiation is ultraviolet radiation.
 26. The method of claim23 wherein the apoptotic stimulus is a cancer chemotherapeutic drug. 27.The method of claim 26 wherein the cancer chemotherapeutic drug isVP-16, cisplatinum or taxol.
 28. The method of claim 23 wherein theapoptotic stimulus is a cellular signaling molecule.
 29. The method ofclaim 23 wherein the apoptotic stimulus is ceramide, a cytokine orstaurosporine.
 30. The method of claim 23 wherein said apoptoticstimulus causes mitochondrial dysfunction.
 31. The method of claim 30wherein said mitochondrial dysfunction is loss of mitochondrial membranepotential.
 32. The method of claim 23, wherein said cell is a cancercell.
 33. The method of claim 32, wherein said cancer cells areglioblastoma or leukemia cells.
 34. A method of promoting apoptosis ofcancer cells, comprising contacting said cells with the antisensecompound of claim
 1. 35. The method of claim 34, further comprising thestep of contacting said cells with a chemotherapeutic agent.
 36. Themethod of claim 35, wherein said chemotherapeutic agent is doxorubicinor dexamethasone.
 37. The method of claim 35, wherein said cancer cellsare glioblastoma cells or leukemia cells.
 38. A method of inhibiting theexpression of bcl-x in human cells or tissues comprising contacting saidcells or tissues in vitro with the antisense compound of claim 1 so thatexpression of bcl-x is inhibited.
 39. A method of sensitizing a cell toan apoptotic stimulus comprising contacting a cell in vitro with theantisense compound of claim
 1. 40. A method of promoting apoptosis ofcancer cells comprising contacting said cells in vitro with theantisense compound of claim 1.